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Genetic analysis of thiamine metabolism

in Saccharomyces cerevisiae

Thesis Submitted for the Degree

of Doctor of Philosophy at the

University of Leicester

By

Kerry Byrne

Department of Genetics

University of Leicester

March 1998

UMI Number: U594540

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Genetic analysis of thiamine metabolism in Saccharomyces cerevisiae

Kerry Byrne

ABSTRACT

A genetic analysis of thiamine metabolism has been carried out in the budding yeast, Saccharomyces cerevisiae. A collection of thiamine auxotrophic mutants were isolated following UV and Ty insertion mutagenesis. The mutations responsible for the auxotrophic phenotypes were characterised to different extents through complementation analysis, molecular cloning and enzyme assays. In total 171 mutants were analysed and all of these have been assigned to complementation groups, genes and/or functions.

Some newly isolated mutations were found to be allelic with the known biosynthetic genes, THI4 and 777/6; others were in the regulatory genes, 777/2 and 777/3; two more defined a new function for the transcription factor, Pdc2p, namely thiamine gene activation. In addition the previously known mutations, thil, thi2, and thi3, were complemented and the sequences of the wild-type 777//, THI2 and THI3 genes were found. From the deduced amino acid sequences roles for the gene products were hypothesised. The Thi2p was found to be homologous with the Gal4p transcription factor due to the presence of a Zn-finger motif; therefore a DNA-binding transcription factor role was proposed for this protein. The Thi3p was found to be homologous to the structural proteins for the enzyme pyruvate decarboxylase. It contains a conserved sequence for TPP binding, the consensus motif having been found in all TPP-dependent enzymes. Therefore it is hypothesised here that Thi3p acts as a "TPP sensor" within the cell, such that deactivation of thiamine-regulated genes is exerted when TPP is bound to Thi3p.

In the case of 7 7 ///, the complementing ORF was found to be a previously characterised gene, ILV2, which encodes the aceto-hydroxy acid synthase (AHAS) enzyme. AHAS catalyses the first step in the parallel biosyntheses of the branched-chain amino acids, isoleucine and valine, using TPP as a cofactor. It was hypothesised that thil encodes a functional AHAS which has a reduced affinity for TPP resulting in a thiamine auxotrophic phenotype, The thil allele was cloned and enzyme assays were carried out which supported this hypothesis. Sequencing analysis and site-directed mutagenesis revealed that die thil phenotype was produced as a result of a single point mutation which caused the conserved amino acid substitution D176E. Hence in this study an amino acid residue potentially important in the binding of TPP to the AHAS enzyme is identified.

ACKNOWLEDGEMENTS

This work was carried out during a three year period on a Research Assistantship funded by the BBSRC.

There were three third year undergraduate project students whose work was included in this thesis: Emma Richards, who carried out tetrad and complementation analysis on ten of the Ty insertion mutants and also cloned the wild-type gene for ty3-l (THI6); Matthew Dean, who isolated the complementing clone for tyl-3 (77/72); and Katie Miles, who isolated the complementing clones for thil and thi3.

There are many people who over the years have helped me along the road to scientific glory. Firstly, Pete Meacock for employing me for (most of) the last six and a half years and for many morale boosting sessions; Annette Cashmore for persuading me to give science another chance after my less than impressive start in a lab; Uta Praekelt who first initiated me into the ways of thiamine. Also, there are the many members of lab F5 both past and present, who have been either a hindrance or a help including: Kath Duffy (for dragging me across to the Redfeam when I wanted to get on with my work!), Bill Gill (for giving me a home), Rob Burrows, Simon E-E (for useful conversations scientific and otherwise) Dickie Hather, Raff Schaffrath, Debs Dawson, Surinder Soond, Dave Walsh, Louise Kew, Emma Richards and Nik Shaw; then there's the present crew Noel Curtis (who hasn't finished yet!), Marcus Marvin (who appreciates the need for Rapid Ramping), Mark McDermott, Glen Palmer, Jane Dickens, Jen Logue and of course Pat Wilson (for all my glassware and media needs) all of whom I can thank for alcoholic stress-busting sessions in many and varied locations as well as lots of laughs along the way.

Finally, I would like to thank my Mum and Dad and the rest of my family for their support and faith in me.

ABBREVIATIONS

ACH Acyl-CoA hydrolaseACS Acetyl-CoA synthetaseADH Alcohol dehydrogenaseADP Adenosine diphosphateAHAS Acetohydroxyacid synthaseAIR Aminoimidazole ribotideALD Acetaldehyde dehydrogenaseALS Acetolactate synthaseARB Asparagine rich boxARS Autonomously replicating sequenceATP Adenosine triphosphateBCAAT Branched-chain amino acid transaminaseBCKD Branched-chain a-keto acid dehydrogenase

bp Base pairsBME p-Mercaptoethanol

CAT Chloramphenicol acetyl transferasecDNA Complementary DNAdATP Deoxyadenosine 5'-triphosphatedCTP Deoxycytidine 5'-triphosphatedGTP Deoxyguanosine 5'-triphosphatedITP Deoxyinosine 5'-triphosphateDNA Deoxyribonucleic aciddNTP Deoxynucleotide 5’-triphosphateDTT DithiothreitoldTTP Deoxythymidine 5'-triphosphateEDTA Ethylenediamine tetra acetic acidEMS Ethyl methanesulphonateFAD Flavin adenine dinucleotide5-FOA 5-fluoroorotic acidHET 4-MethyJ-5-p-hydroxyethylthiazolea-HIC a-HydroxyisocaproateHMP 2-Methyl-4-amino-5-hydroxymethylpyrimidineIAA Isoamyl alcoholkb KilobaseskDa KilodaltonsKGD a-Ketoglutaratea-KIC a-Ketoisocaproate

LB Luria BertanimRNA Messenger ribonucleic acidnt NucleotidesONPG 0-Nitrophenyl-|3-D-galactopyranoside

ORF Open reading framePCR Polymerase chain reactionPDC Pyruvate decarboxylasePDH Pyruvate dehydrogenasePEG Polyethylene glycolPi Inorganic phosphatePP PyrophosphateRDA Recommended daily amountRNA Ribonucleic acidSD Synthetic definedSDS Sodium dodecyl sulphateTCA Tricarboxylic acidTK TransketolaseTMP Thiamine monophosphateTPK Thiamine pyrophosphokinaseTP Thiamine phosphateTPP Thiamine pyrophosphateTPS Thiamine phosphate synthaseT-rAPase Thiamine-repressible acid phosphataseTTC 2,3,5-Triphenyltetrazolium chlorideUAS Upstream activation siteUV UltravioletW/V Weight / volumeYPD Yeast peptone dextroseYLE Yeast lytic enzymeYGSC Yeast Genetic Stock Centre

TABLE OF CONTENTS

CHAPTER ONEIntroduction...........................................................................................................................11 .1 B a c k g r o u n d ........................................................................................................ 11.2 Thiamine pyrophosphate as a cofactor...............................................................11.3 T P P -d ep en d en t en zy m es..............................................................................21.3.1 Pyruvate decarboxylase........................................................................................21.3.2 Pyruvate dehydrogenase.......................................................................................31.3.3 Pyruvate oxidase ...................................................................................................... 41.3.4 Transketolase................................................................................................................. 41.3.5 Aceto Hydroxy Acid Synthase...........................................................................51.3.6 a-ketoglutarate dehydrogenase..................................................................................... 61.4 Comparisons between different TPP enzymes................................................ 71.4.1 Catalysis by TPP ............................................................................................................71.4.2 TPP b ind ing ............................................................................................................91 .5 B io s y n t h e s is ..................................................................................................... 101.5.1 Elucidation of the biosynthetic pathway .......................................................... 101.5.2 Origins of the precursors.......................................................................................... ..111.5.2.1. Hydroxyethy 1 thiazole........................................................................................... 111.5.2.2 Hydroxymethyl pyrimidine...................................................................................... 131.5.3 Genetics of thiamine biosynthesis in bacteria............................................................. 141.5.4 Genetics of thiamine biosynthesis in yeast................................................................. 151.5.4.1 T H I 4 ................................................................................................ 151.5.4.2 7 7 /7 5 ......................................................................................................................171.5.4.3 T H I 6 .......................................................................................................................181.5.4.4 TH I80 ........................................................................................................................ 191.6 The thiamine transport pathw ay........................................................................ 191.6.1 Proteins involved in transport..........................................................................201.6.2 Genes involved in thiamine transport......................................................................... 211.6.3 Transport of precursors.....................................................................................221.7 Regulation of thiamine metabolism................................................................... 231.7.1 The thi2 and thi3 mutations.........................................................................................231.7.2 Regulation of biosynthesis.......................................................................................... 241.7.3 Regulation of the transport pathway...........................................................................251.7.4 Regulation in other organisms.....................................................................................251.8 Aims of the project................................................................................................. 26

CHAPTER TWOMaterials and M ethods...................................................................................................282.1 S tra in s and p la sm id s ..............................................................................282.1.1. Yeast strains (Saccharomyces cerevisiae)................................................................. 282.1.2 Bacterial strains (Escherichia coli)......................................................................292.1.3 Plasmids and vectors......................................................................................... 292.2 Growth media and conditions..............................................................................302.2.1. Bacterial m edia...................................................................................................302.2.2. Yeast media and growth conditions...........................................................................302.3 M a n ip u la tio n s in y ea st ..........................................................................302.3.1 Low efficiency transformation.....................................................................................302.3.2 High efficiency transformation................................................................................... 312.3.3 Small scale plasmid preparations from S. cerevisiae................................................. 312.3.4 Plasmid curing.............................................................................................................. 322.3.5 Preparation of highly purified genomic DNA.............................................................322.3.6 Gene d isruption ................................................................................................... 322.3.7 Gap repair..................................................................................................................... 332.3.8 Making Y02587 ura3 ...............................'...................................................................332.3.9 Making 058-M5 ura3 ...................................................................................................332.4 Manipulations in E. coli....................................................................................... 342.4.1 Transformation.............................................................................................................342.4.2 Small scale plasmid preparations................................................................................ 342.4.3 Large scale plasmid preparations................................................................................ 342.4.4 Bacterial transposon mutagenesis.......................................................... 342.5 DNA m anipulations................................................................................................ 352.5.1 Restriction enzyme digests.......................................................................................... 352.5.2 DNA agarose gel electrophoresis................................................................................ 352.5.3 Isolation of DNA fragments............................................................................352.5.4 DNA dephosphorylation.................................................................................... 352.5.5 DNA ligation................................................................................................................. 352.5.6 Southern blotting.......................................................................................................... 352.5.7 Hybridisation with a 32p radiolabelled probe.................................................. 362.5.8 Hybridisation with a non-radioactive probe................................................................362.5.9 DNA sequencing.......................................................................................................... 362.5.10 Polymerase chain reaction......................................................................................... 372.6 M utagenesis............................................................................................................... 382.6.1 UV mutagenesis............................................................................................................382.6.2 Ty insertion mutagenesis............................................................................................. 382.6.3 Site-directed m utagenesis.................................................................................. 39

vi

2.6.4 Nystatin enrichment................................................................................................... 392.7 A n a ly sis o f m u ta n ts .................................................................................392.7.1 Dominance / recessiveness test against wild-type......................................................392.7.2 Sporulation of diploids and dissection of tetrads....................................................... 392.8 Enzyme assays..........................................................................................................402.8.1 Preparation of permeabilised cells...............................................................................402.8.2 Acetohydroxyacid synthetase (AHAS) assay.............................................................402.8.3 Assay for P-galactosidase activity in liquid culture..........................................41

CHAPTER THREEIsolation and characterisation of thiamine auxotrophs.....................................423.1 In troduction ............................................................................................................... 423.2 Mutagenesis..................................................................................................................... 433.2.1 UV mutagenesis...........................................................................................................433.2.2 Ty mutagenesis............................................................................................................ 433.3 Genetic analysis............................................................................................................. 453.3.1 Dominance / recessiveness test...................................................................................453.3.2 Meiotic segregation analysis.......................................................................................453.3.3 Cosegregation of His+ and Thi' alleles...................................................................... 463.4 Complementation analysis.............................................................................................. 473.5 Growth phenotypes................................................................................................ 483.6 Further analysis of uv2 and uv3..................................................................................... 493.7 Discussion........................................................................................................................ 50

CHAPTER FOURMolecular cloning of the THI 6 gene........................................................................ 524.1 In troduction ............................................................................................................... 524.2 Molecular cloning of the wild-type UV4 and TY3-1 genes.......................................... 524.2.1 Confirmation that the library plasmids carry the complementing genes...............534.2.2 Characterisation of the library clones.......................................................................... 544.3 Allelism of uv4 and ty3 -l with th i6 .....................................................................544.3.1 Comparison of the restriction maps............................................................................ 554.3.2 PCR analysis ........................................................................................................554.3.3 Southern blot analysis of the mutant strains compared with wild-type.................... 554.4 Localisation of the Ty element within the Ty3-1 genome............................................. 564.5 Discussion........................................................................................................................ 56

CHAPTER FIVECharacterisation and genetic analysis of the th i l m utation...................... 585.1 In troduction ................................................................................................................585.2 Genetic analysis of t h i l ...................................................................................................585.2.1 Dominance / recessiveness test and complementation analysis................................ 585.2.2 Cloning the wild-type TH11 gene............................................................................... 585.2.3 Allelism of thil and ilv2 ...............................................................................................595.2.4 Disruption of the ILV2 locus............................................................................ 605.2.5 Nutritional characteristics of thil and ilv2 mutants.................................................... 615.2.6 Segregation of ilv2 and thil alleles..............................................................................615.3 Cloning the thil allele......................................................................................................625.4 Acetolactate synthetase assays............................................................................. 635.5 Mutation mapping by fragment exchange.......................................................................645.6 Characterisation of the thil mutation..............................................................................645.7 Site-directed mutagenesis................................................................................................655.8 Confirmation of phenotype for the site directed mutant................................................665.9 Confirmation of the Stul site in the genome of the thil strain....................................... 665.10 Discussion..................................................................................................................... 67

CHAPTER 6Molecular cloning of the THI2 and THI3 gen es.................................................... 696.1 In troduction ..................................................................... 696.2 Molecular cloning of TH12..............................................................................................696.2.1 Analysis of p K M l.............................................................................................696.2.2 Identifying the THI2 ORF..................................................................................706.3 Disruption of the YBR240c locus.......................................................................706.4 Thi2p is a Zn-finger DNA binding protein.........................................................716.5 Allelism of thi2 and tyl-3 ................................................................................................716.5.1 Segregation of thi2 and ty l-3 ...................................................................................... 716.5.2 Southern blot analysis of Tyl-3.................................................................................. 716.5.3 Crossing the thi2 disruption strain with tyl-3 ............................................................ 726.6 Molecular cloning of THI3..............................................................................................726.6.1 Analysis of pKM 2.............................................................................................726.6.2 Identifying the THI3 ORF..................................................................................736.7 Disruption of the YDL080c locus................................................................................... 736.8 Thi3p is a PDC-like protein................................................................................746.9 Growth phenotypes of disruption strains and the original mutants...............................746.10 D iscussion ................................................................................................................ 74

CHAPTER SEVENGeneral d iscussion ..........................................................................................................767.1 Auxotrophic mutations in biosynthetic genes..................................................... 767.2 Mutation in a TPP-dependent enzyme............................................................................ 777.3 Mutations in regulatory genes........................................................................................ 797.3.1 The YDL080c O R F....................................................................................................817.4 Futher w ork ..................................................................................................................... 82REFERENCES...................................................................................................................84

CHAPTER ONE

Introduction

1.1 BackgroundVitamin B i (thiamine), previously known as aneurin, is found naturally in

microorganisms such as bacteria and yeast, and plants; all of which synthesise the vitamin de novo. Discovered in a fraction of rice polishing in the search for a cure for beri-beri, thiamine was the first vitamin to be identified and was isolated from yeast in 1932. Its structure was determined soon afterwards and the role of thiamine pyrophosphate (TPP) as a cofactor was subsequently proposed (reviewed by Begley, 1996).

Human inability to synthesise thiamine means that it is an essential dietary requirement (RDA = 1.4mg) and nowadays white bread and many breakfast cereals are supplemented with this essential vitamin. For this purpose it is obtained almost entirely from chemical synthesis, as there is no rich natural source of thiamine, not even yeast which is the primary source of vitamins B2 and B 12 (Haj-Ahmad et al., 1992). Chemical synthesis used

for food supplementation is in excess of 2000 tons annually.Dietary deficiency of thiamine can lead to disorders of the peripheral nervous system

eg. beri-beri, or diseases affecting the central nervous system. A striking neuropsychiatric disorder known as Wemicke-Korsakoff syndrome or Wernicke's encephalopathy causes paralysis of eye movement, abnormal stance and deranged mental function due to lack of thiamine in the diet of sufferers. These symptoms may arise from a defect of the TPP- dependent enzyme, transketolase (TK), which binds the cofactor less avidly than a normal TK. However, there is speculation as to whether there is an inborn TK abnormality in Wemicke-Korsakoff patients (Blansjaar et al., 1991).

1.2 Thiamine pyrophosphate as a cofactorThiamine pyrophosphate (TPP), the biologically active form of vitamin B 1, is a

cofactor for enzymic reactions that involve the cleavage of a C-C bond adjacent to a carbonyl group. It was first found to act as a coenzyme for yeast pyruvate decarboxylase (PDC) in 1937 (reviewed in Leder, 1975). Soon afterwards it was shown that TPP was required for all enzymic reactions involving cleavage of pyruvate, including the formation of acetoin, formate, acetyl phosphate, acetyl CoA and COr, thus pyruvate cannot be metabolised without TPP. By 1953 the coenzyme had also been shown to be required for cleavage of D- xylulose-5-phosphate by the enzyme transketolase (TK) (reviewed by Goodwin, 1963). To date, at least fourteen enzymes have been found to require TPP and a divalent cation for catalysis (Schenk et al., 1997).

1

The free coenzyme is able to carry out decarboxylation of 2-oxo acids such as pyruvate, but the reaction rate can be increased some 1012 fold when TPP is complexed with a PDC enzyme (Alvarez et al., 1991). Identifying specific interactions of a coenzyme with the protein is therefore crucial to understanding how it exerts catalytic activity. To this end the three dimensional X-ray crystallographic structures of three important enzymes utilising TPP as a cofactor have been resolved: PDC from Saccharomyces cerevisiae (Dyda et al.,1993); pyruvate oxidase (POX) from Lactobacillus plantarum (Muller & Schultz, 1993); and TK also from S. cerevisiae (Lindqvist et al., 1992; Nilsson et al., 1993).

A conserved consensus sequence has been found which is present in all TPP-binding enzymes so far identified. Alignment of the amino acid sequences of various TPP-dependent enzymes including, PDC, POX, TK, pyruvate dehydrogenase (subunit E l) (PDH-E1), acetohydroxyacid synthase (AHAS) and a-ketoglutarate dehydrogenase (KGD) from yeast,

Escherichia coli, humans and plants revealed a common conserved sequence, GDGX(25- 28)NN now termed the "TPP-binding motif' (Hawkins, 1989). Figure 1.1 shows the aligned sequences and motif.

1.3 TPP-dependent enzymesThe several classes of enzymes which use TPP as a cofactor can be grouped by their

activitiesi) decarboxylation of pyruvate, PDC and AHAS;ii) oxidative decarboxylation of pyruvate; the El components, of PDH and KGD (PDH-E1 and KGD-E1) and POX;iii) transfer of two carbon units from ketones to aldoses; TK.Five of these enzymes have been found in S. cerevisiae: PDC, AHAS, PDH-E1, KGD-E1 and TK, along with three PDC-like ORFs of unknown function - YDR380w, YEL020c and YDL080c.

1.3.1 Pyruvate decarboxylaseTPP-dependent PDC is a key enzyme in the fermentation process following

glycolysis. It irreversibly catalyses the decarboxylation of pyruvate to acetaldehyde and CO2

(Figure 1.2). In yeast the enzyme is encoded by three separate genes, PDC1, PDC5 and PDC6, although more than 90% of the total protein is encoded by the PDC1 gene. The protein products are very similar with the Pdc5p and Pdc6p enzymes showing 88% and 84% identity to Pdclp. The PDC1 and PDC5 genes are under regulation by glucose whilst the PDC6 gene is not normally actively transcribed; the PDC5 gene is subject to further regulation by the Pdclp (Hohmann et al., 1996) and thiamine (from work originating in this study but continued by Richards, 1996). Both a pdclA pdcSA double mutant and a pdclA pdc5A pdc6A triple mutant grow very poorly on glucose medium: this is not solely due to

2

Zmpdc APERRNILMVGDGS - FQLTAQEVAQMVR - LKL - P V IIF L IN N Y - GYTIEVM

Scpdcl DPKKRVILFIGDGSL-QLTVQEISTMIRWGLK-PY-LFVLNND-GYTIEKL

Ntsura RPDEVWDIDGDGS-FIMNVQELATIKVENL--PVKIMLLNNQ-HLGMWQ

Scilv2 KPESLVIDIDGDAS - FNMTLTEL S S-AVQAGT - PVKILILNNEE - QGMVTQ

Ecilvi LPEETWCVTGDGSI-QMNIQELST-ALQYEL-PVLWNLNNR-YLGMVKO

Ecpoxb EPERQWAMCGDGG- F SMLMGDFLS - WQMKLPVKIW FNNS - VLGFVAM

HuE 1 ap GKDEVCLTL YGDGAANQGQIFEAYNMAALWKL - PC IFICENNY - GMGT S VE

B sE la GKKAVAITYTGDGGTSQGDFYEGINFAGAFKA-PAIFWQNNR--FAISTP

EcaceE TSKQTVYAFLGDGEMDEPESKGAITIATREKL-DNLVFVINCNLQR-LDGP

Sckgdl LLHGDA-AFAGQGW-YETM-GFLTL-PEYSTGGTIHVITNNQIGFT-TDP

EcsucA TIHGDA-AVTGQGW-QETL-NMS-KARGYEVGGTVRIVINNQVGFTTSNP

H llElab NANRW ICYFGEGAASEGDAHDGFNFAATLEC-PIIFFCRNN--GYAISTP

O xElab NANRWICYFGEGAASEGDAHAGFNFAATLEC-PIIFFCRNN--GYAISTP

R aE lab NANQWICYFGEGAASEGDAHAGFNFAATLEC-PIIFFCRNN--GYAISTP

PpbkdAl GDTKIASAWIGDGATAESDFHTALTFAHVYRA-PVILNWNNQ--WAISTF

Ysmdas IITNKVYCMVGDACLQEGPALE SISLAGHMGLDNLIVL YDNNQVCCDGSVD

Rcrcfp Q P VGDTIA11GDG S IT AGMA YE ALNHAGHL K - - S RMF VILNDND - M S IA P P

P 1 ------------------a -------------------- t - - p ----- 1 ------- a -------

Figure 1.1

The TPP-binding consensus sequence.

Alignment of the amino acid sequences of various TPP-dependent enzymes, adapted from Hawkins et al. (1989). Highlighted are the conserved residues forming the putative "TPP- binding motif'. The predicted secondary structure is given underneath.The enzymes include:- PDC from Zymomonas mobilis and S. cerevisiae - Zmpdc and Scpdcl, respectively; ALS from Nicotiana tabacum, S. cerevisiae and E. coli - Ntsura, Scilv2 and Ecilvi, respectively; POX from E. coli - EcpoxB; PDH-E1 from Human, Bacillus stearothermophilus and E. coli - H uE lap, B sE la and EcaceE, respectively; a-keto-

glutarate dehydrogenase from S. cerevisiae and E. coli - Sckgdl and EcsucA, respectively; branched chain 2-oxo acid dehydrogenase complex from Human, Ox, Rat and Pseudomonas putida - HuE la b , O xE lab , R aE lab and PpbkaA l, respectively; formaldehyde

transketolase (dihydroxyacetone synthase) from Hansenula polymorpha - Ysmdas; and an unidentified ORF from R. capsulata - Rcrcfp.

GLUCOSE

Glycolysis

PDCPYRUVATE

PDH

ADHACETALDEHYDE -----► ETHANOL

+ C 0 2

ALD

ACETYL-CoAACS

ACETATE

tTCA CYCLE

Figure 1.2

Pvruvate at the branchpoint of sugar metabolism

Pyruvate can be metabolised to acetyl coenzyme-A by pyruvate dehydrogenase (PDH). It is subsequently oxidised to carbon dioxide and water via the TCA cycle. Alternatively, during fermentation, pyruvate is converted to acetaldehyde by pyruvate decarboxylase (PDC) and then to ethanol by alcohol dehydrogenase (ADH). Theoretically, the acetaldehyde can also be converted to acetate and subsequently to acetyl CoA by acetaldehyde dehydrogenase (ALD) and acetyl-CoA synthetase (ACS), respectively.

glucose repression of the TCA-cycle but also because of a lack of cytosolic acetyl-CoA (produced from acetaldehyde) since yeast cells are unable to export this compound from the mitochondria. The transcriptional activator Pdc2p enhances expression of the PDC1 gene whilst being essential for expression of the PDC5 gene (Hohmann, 1993; Raghuram et al.,1994). For a review see Hohmann & Meacock (1998).

The PDC enzyme is a tetramer of identical 60kDa subunits each consisting of three domains - a , p, y. At physiological pH, around pH 6, four TPP and Mg2+ ions are bound to

the PDC tetramer in a quasi-irreversible manner. In both yeast and Zymomonas mobilis it has been found that the cofactors dissociate from the holoenzyme at pH values above 8.0 and recombine with the apoenzyme in a time-dependent manner at pH values below 6.5 (Diefenbach & Duggleby, 1991).

All known PDCs - with the exception of that from Z. mobilis - are subject to allosteric activation by the substrate or a substrate analogue and are inhibited by inorganic phosphate; high pyruvate and low Pi conditions are associated with high glycolytic activity. Allosteric activation is thought to occur via binding of cofactors to the regulatory p-domain

which forms interactions that hold the dimer-dimer assembly together (Lu et al., 1997). Cooperative kinetics for activation by the cofactors, TPP and Mg2+, have been demonstrated in the PDC enzyme from Z. mobilis (Diefenbach and Duggleby, 1991). However, the possibility of an association between the two cofactors before binding is discounted by the large difference in rate constants for association of each cofactor; a six-fold difference for the first association and a 13-fold difference for the second. Thus the cofactors must associate separately.

1.3.2 Pyruvate dehydrogenaseThe multi-enzyme complex of PDH (PDHc) carries out the oxidative decarboxylation

of pyruvate, within the mitochondria of eukaryotes. PDHc is central to carbohydrate metabolism in both prokaryotes and eukaryotes, producing acetyl CoA the initial substrate for the respiratory pathway (Figure 1.2). It is also involved in producing biosynthetic intermediates. The complex carries out the overall reaction :

Pyruvate + CoA + NAD+ —> Acetyl-CoA + CCH + NADH +H+

The complex consists of three components: PDH (E l), dihydrolipoamide transacetylase (E2) and lipoamide dehydrogenase (E3); stoichiometry of the polypeptides is 24 : 24 : 12. In yeast, PDH contains a regulatory subunit X, called Pdxlp. PDH-E1 itself consists of two proteins - Pdalp and Pdblp encoded by the PDA1 and PDHfil genes in S.

cerevisiae. It carries out the first part of the reaction above:

Pyruvate + El-TPP-Mg2+ —> El-hydroxyethylidene-TPP-Mg2+ + CCb

3

The hydroxyethylidene intermediate is converted to acetyl dihydrolipoamide and then dihydrolipoamide and acetyl CoA by the E2 enzyme; finally NADH is produced from the dehydrogenation of the lipoamide by the E3 enzyme. Mutants lacking Pdalp display a partial requirement for leucine, perhaps due to the requirement of isopropylmalate synthase (the first enzyme in leucine biosynthesis) for mitochondrial acetyl-CoA (reviewed by Hohmann and Meacock, 1998).

The PDH-E1 protein is a tetramer of (X2 P2 configuration. The cofactor is optimally

bound to PDH at pH 7.0 - 7.5 and appears to be rapidly reversible, whilst in PDC it is optimally bound at pH 6.0 and is removed at pH8.0 - 8.5.

As is the case for the PDC enzyme, PDH is subject to substrate and cofactor activation, being activated by TPP and pyruvate whilst it is inhibited by NADH, Acetyl-CoA and pyruvate analogues. The wild-type enzyme undergoes a fast binding step, followed by a slow (50-100 secs) isomerisation before steady-state product is released. The lag phase of product formation when TPP and pyrvuate was added to the apoenzyme was abolished in the mutant proteins with impaired TPP folds (Jordan et al., 1996). It was found that the TPP- dependent lag phase was not related to pyruvate catalysis but was a result of slow activation of PDH, specifically by the rate of TPP binding. This probably involves stepwise, cooperative binding of two TPP-Mg2+ molecules to the dimer indicating that the TPP-Mg2+ slowly penetrates the binding pocket and induces an "activated fit" (Yi et al., 1996). The idea of cooperativity of binding is encouraged by the fact that TPP binds to TPP-dependent enzymes at the interface of identical subunits.

1.3.3 Pyruvate oxidasePyruvate oxidase uses TPP as a coenzyme to catalyse the oxidative decarboxylation

of pyruvate through various steps, to produce hydrogen peroxide and acetyl phosphate, an energy-storage metabolite which can be used by acetate kinase to convert ADP to ATP (Muller and Schultz, 1993). It is important in the aerobic growth of lactobacteria. The enzyme is encoded by the poxB gene in E. coli.

The three-dimensional crystallographic structure of the enzyme showed that it is a homotetrameric enzyme with the monomeric polypeptides consisting of three domains - core, FAD-binding and TPP-binding, the core domains forming the centre of the tetramer. Each domain consists of a six-stranded parallel p sheet surrounded by a helices, as in all TPP-

binding enzymes studied (Muller and Schultz, 1993).

1.3.4 TransketolaseIn general, TK enzymes catalyse the transfer of a two carbon unit from a ketose to an

aldose acceptor within the pentose phosphate pathway (Nikkola et al., 1994). Together with transaldolase, TK forms a reversible metabolic link between the glycolytic and pentose phosphate pathways, facilitating the cycling of ribose-5-phosphate and other glycolytic

4

intermediates. The functional enzyme is a homodimer which binds two TPP molecules and two Mg2+ or Ca2+ ions reversibly, each monomer being approximately 74Kd (Sundstrom et al., 1993) TK is encoded by two genes in S. cerevisiae TKL1 and TKL2 and the resulting proteins are 71% identical (Schaaff-Gerstenschlager et al., 1993; Sundstrom et al., 1993). Although two TK proteins have been isolated from both E. coli and S. cerevisiae it is not clear whether these have distinct functions however these enzymes display a wide range of substrate specificity, being able to utilise 3-C to 7-C sugars. In plants TK also plays a vital role in the Calvin cycle, using glyceraldehyde-3-phosphate as a substrate. TK enzymes may have become adapted to this breadth of substrate recognition, at the expense of efficiency, turnovers having been shown to be rather low.

Human TK is 24% identical to the yeast enzyme and is 75% identical for the residues that interact with TPP (Singleton et al., 1996). Phylogenetic studies were carried out on 22 transketolase sequences from 17 different organisms and the four phlya - yeasts, mammals, bacteria and plants. A high degree of conservation in function was found and some important residues, including 50 totally invariant ones and 24 which only differ in the mammalian sequences, indicating that they all derive from a common ancestral gene which has evolved slowly (Schenk et al., 1997). A highly conserved stretch was found comprising Thr481 to Asp503 which was designated the "transketolase m otif. This region contains residues crucial to subunit dimerisation thereby forming the site for binding of the thiazolium and pyrimidine rings of TPP, as well as substrate binding (Nikkola et al., 1994).

1.3.5 Aceto Hydroxy Acid SynthaseAceto-hydroxy acid synthase (AHAS) is the enzyme responsible for the first step in

the parallel synthesis of the branched chain amino-acids isoleucine, valine and leucine. The reactions catalysed by AHAS convert pyruvate and a-ketobutyrate to oc-

acetohydroxybutyrate, an intermediate in the biosynthesis of isoleucine, and two pyruvates to acetolactate, an intermediate in the biosynthesis of valine. Both reactions involve decarboxylation of pyruvate catalysed by the cofactor TPP, as in the PDC and PDH enzymes. The synthesis of acetolactate from pyruvate displays Michaelis-Menten kinetics and valine is a non-competitive inhibitor of the enzyme (Magee & Robichon-Szulmajster, 1968).

AHAS is a complex dimer with a a2p2 structure and in E. coli has three isozymes,

each with a similar structure but different kinetic and physiological functions. These are encoded by ilvBN (AHAS I), ilvGM (AHAS II), and ilvIH (AHAS III) (Weinstock et al., 1992). Isozymes I and III are sensitive to feedback inhibition by valine, whereas isozyme II is not (De Felice et al., 1982). The larger a subunits are approximately 60 kDa, have high

sequence similarity and have been found to carry out the catalytic functions. The smaller more heterogeneous P subunits (9 - 17kdal) encoded on the same operon as the larger

subunits distal to the promoter, carry out the regulatory functions, conveying feedback

5

inhibition by valine on the enzyme. They have also been shown to play an important role in enhancing the activity of the large subunits by inducing a catalytic ally competent conformation which stabilises the transition state.

Despite the similarity of the large subunit in both their amino acid sequences (over 40% identity) and functions, each small subunit interacts specifically with its larger counterpart, perhaps reflecting their different physiological roles. It has been suggested that AHAS I enables a bacterium to cope with poor carbon sources which leads to low endogenous pyruvate levels; in contrast AHAS II and m fail to produce adequate amounts of acetolactate when pyruvate levels are low (on non - glucose media) (Barak et al., 1987).

In S. cerevisiae AHAS is encoded by a single gene 1LV2 located on the right arm of chromosome XIII. It is homologous to the large catalytic subunits of E. coli AHAS II and AHAS III, with 40% of the amino acids being identical (Falco et al., 1985). Surprisingly, the yeast enzyme is as similar to each of the bacterial proteins as they are to each other, apart from a putative NH2-terminal mitochondrial signal sequence present in the yeast protein. A gene encoding the regulatory subunit of AHAS has recently been identified in S. cerevisiae, ILV6 (YCL09c). This polypeptide has been shown to confer valine sensitivity to the enzyme and was found to be homologous with a number of prokaryotic small regulatory subunits (Cullin et al., 1996). A homologue of ILV2 has also been isolated from the fission yeast Schizosaccharomyces pombe - ilvl. It is 57.1% identical to the S. cerevisiae gene and was isolated after screening a S. pombe genomic library with 32p labelled DNA of the ILV2 ORF. The ilvl gene has been shown to functionally complement a S. cerevisiae ilv2 mutant strain, confirming its role as a structural gene for AHAS.

Genes encoding AHAS have been widely studied in plants as well as yeast because it is the site for resistance to the herbicide sulfometuron methyl. A number of resistance alleles have been isolated in both yeast and plants (Chaleff & Mauvais, 1984; Falco & Dumas, 1985; Hattori et al., 1992). Brewers are also interested in the ILV2 gene since one of the products of AHAS action, acetolactate, can be converted non-enzymically to diacetyl, the substance responsible for the off-flavour in beer. A reduction in AHAS activity, brought about by a mutation in the ILV2 gene, or an increase in acetohydroxyacid reductoisomerase activity, the product of the 1LV5 gene which converts acetolactate to dihydroxyisovalerate, would therefore be helpful in reducing the build up of diacetyl and the likelihood of off- flavour beer (Gjermansen et al.. 1988).

1.3.6 a-ketoglutarate dehydrogenaseThe TCA cycle enzyme, KGD, carries out the oxidative decarboxylation of a -

ketoglutarate and like PDHc, acts within a multienzyme complex in the mitochondria. It carries out the overall reaction:

a-ketoglutarate + CoA + NAD+ —> succinyl CoA + CO2 + NADH +H+

6

Like PDHc, KGDc comprises two specific subunits E l and E2, with the E3 subunit, lipoamide dehydrogenase being common to both complexes (Stryer, 1988). In S. cerevisiae KGD-E1 is encoded by a single enzyme, KGD1 expression of which is under the regulation of glucose (Repetto & Tzagoloff, 1989).

1.4 Comparisons between different TPP enzymesA comparison of the crystallographic structures of the PDC, POX and TK enzymes

has been carried out (Muller et al., 1993). It was found that despite considerable differences in quaternary structure and lack of overall sequence homology, TPP binds to the three enzymes in a very similar fashion. A number of amino acid side chains involved in cofactor binding and activation are conserved between the proteins; ten are situated in the pyrophosphate (PP)-binding domain and two in the domain where the pyrimidine ring binds.

In the PDC tetramer of identical 60kDa subunits, each comprising a , (3, y, domains, the a and y domains are similar to each other and are involved in the binding of the substrate,

the coenzyme and the divalent cation. Moreover they have the same fold as the corresponding domains of TK and POX. The function of the (3 subunit is thought to be

regulatory, being involved in substrate activation.From the phylogenetic studies carried out amongst TKs from different organisms

(Schenk et al., 1997) 50 invariant residues were found. These include, His30, His69, His103 and His263 which form part of the cluster of His residues previously predicted to be involved in substrate binding (Nikkola et al., 1994), along with His481, which is substituted only in mammals to Gin. The other conserved residues include Asp157 (D) and Asn185 (N) from the GDGX(2 5 -2 8 )NN motif; the two Gly (G) residues are not invariant but they are highly conserved, being present in twenty one (different) sequences out of the twenty two.

1.4.1 Catalysis by TPPEnzymatic thiamine catalysis proceeds through two common intermediates,

irrespective of the specific reaction: initially, deprotonation of the highly acidic C-2 atom of the thiazolium ring creates a carbanion and an ylide is formed: then the substrate undergoes a nucleophilic attack by the ylide cleaving the adjacent carbon-carbon bond and yielding an a-

carbanion.Studies with TPP analogues have shown that atoms other than the reactive C-2 on the

coenzyme molecule are essential for catalysis, such as the NT atom and the 4'-NH2 group of the pyrimidine ring (Figure 1.3) (Golbik et a l, 1991). Until recently the base responsible for the abstraction of the thiazole C-2 proton was unknown, although 4'-NH2 was suspected. From the three-dimensional crystallographic structure of TK it was revealed that no enzyme residue was near enough to the C-2 atom of the TPP molecule to carry out

7

0 oII II

H O P O P O

1 iOH OH

PYROPHOSPHATE

2 CH

1’N

3

THIAZOLE PYRIMIDINE

Figure 1.3

Schematic diagram of the thiamine pyrophosphate molecule.

deprotonation. However, after refining the structure to 2.0A resolution it was shown that the bound TPP molecule had a different conformation from the free molecule, importantly bringing the reactive C-2 atom of the thiazolium ring into closer contact (3.15A) with the 4'- NH2 group of the pyrimidine ring (Nikkola et al., 1994). Also studies on the PDC enzyme have shown that the 4'-NH2 group is deprotonated ready for acceptance of a hydrogen atom (Arjunan et al., 1996), reinforcing the idea that it is involved in deprotonation of the thiazolium C-2.

A mechanism for cofactor-assisted proton abstraction of C-2, in which the hydrogen bond between Glu418 and the N l' atom of the pyrimidine ring is essential was proposed (Figure 1.4). The neutral form of Glu418 donates a proton to the NT while a base, His481, abstracts a proton from the 4'-NH2 to yield an imine. Protonated His481 can transfer its proton to bulk solvent. This process is then reversed with C-2 acting as the proton donor resulting in the formation of a carbanion at C-2 (Lindqvist et al., 1992). This mechanism for generating the C-2 carbanion is thought to be common to all TPP-dependent enzymes. However the proposed role of His481 is questionable since mammalian transketolases contain a glutamine at the equivalent position. A mutation of Glu418 in yeast TK to Gin or Ala markedly reduced enzymatic activity (Wikner et al., 1994) indicating its importance. Contentiously, however, it has been suggested-that since neither of these mutants appeared to be totally inactive then the hydrogen bond between Glu418 and N l' is not absolutely critical for catalysis; this is countered by the following evidence:1) The Glu418 residue found in TK is invariant throughout TPP-dependent enzymes.2) Experiments using TPP analogues (Golbik et al., 1991) showed that the hydrogen-bond interaction was essential for the fast dissociation of the C-2 proton (Kem et al., 1997).3) Using the TPP analogues Nl'-pyridyl and N3’-pyridyl it was found that although the analogues bound to the TK enzyme in a very similar fashions to TPP, only the N l’-pyridyl analogue was catalytically active. This analogue contained the N l' atom whilst N3'-pyridyl had a carbon atom substituted at this position. Therefore it was concluded that the lack of a hydrogen bond at N l', rather than a defect in coenzyme binding conformation, caused the loss of catalysis (Konig et al., 1994).4) The importance of the hydrogen bond between the Glu51 residue (the equivalent of the TK Glu418) and the N l' atom of the pyrimidine ring in recombinant PDC was tested by substituting Glu51 with residues which form either a weak hydrogen bond or no hydrogen bond. Gin and Ala, respectively. The Gin mutant protein displayed wild-type kinetics for TPP binding but only 0.04% PDC activity plus a thirty three fold decrease in substrate binding affinity (Nikkola et al., 1994). The Ala mutant, on the other hand, was inactive and displayed slow binding kinetics of TPP and a high dissociation constant, implying that interaction between the Glu51 and the N l' atom of the pyrimidine ring of TPP is indeed necessary for catalysis (Muller et al., 1993).

GIu418 Glu418

RH-

His481

HO O QH3 o - o

R ■>

NH

hnC / /B

His481

C“H-N

His481

HB

(

Figure 1.4

Mechanism of proton abstraction at the C-2 carbon atom of TPP.

B denotes an enzymic base or a water molecule.Taken from Lindqvist et al., 1992. Amino acid numbers are from TK.

Glu418

- O

Another residue important for catalysis is the invariant His103 in TK; substitution with Ala, Asn or Phe residues, caused no structural changes but reduced activity to 4.3%, 2.4% and 0.1%, respectively. The Ala and Asn substitutions led to a weaker binding for the donor substrate (xylulose-5-phosphate) but not the acceptor substrate. It was therefore concluded that His103 facilitates catalysis (but is not essential for it) by binding the C-l hydroxyl group of the donor substrate and stabilising the intermediate (Wikner et al., 1995). In human TK the function of the equivalent residue, His110, was examined (Singleton et al., 1996). It was proposed that the residue in the human TK is involved in the abstraction of a proton from the 4-NH2 group of the pyrimidine moiety along with another residue, Gin428 which stabilises the orientation of a water molecule. The equivalent residue to Gin428 in all other TPP-dependent enzymes is the conserved His481 and, as mentioned above, it is this residue rather than the His103, which is thought to mediate the proton abstraction in these other non-human TK enzymes. However mutating the Gin428 residue to a His in the human TK resulted in a loss of activity of approximately 60%, equivalent to the loss seen in a Ala or Asn mutant (Singleton et al., 1996). Therefore it was concluded that the roles of the conserved residues are different in the human TK compared to other enzymes and that this may reflect differences in repertoires of potential substrates between the different species.

1.4.2 TPP bindingSince the crystallographic structure of the three TPP-dependent enzymes has been

elucidated, analysis of the mechanism for coenzyme binding has become possible. In TK TPP binds between the amino-terminal domain of one subunit and the middle domain of the second. The coenzyme pocket has an opening just wide enough to admit the sugar phosphate substrate (Nikkola et al., 1994). It is shielded from the solvent except for a small part of the thiazolium ring which exposes the reactive C-2 atom making it accessible to the substrate. Two short strands from the (3-sheet form a hairpin which serves to protect the coenzyme

from the solvent. Removal of the coenzyme causes the two loop regions to become more flexible allowing access to the shielded binding site. One strand, which binds TPP directly, was seen to be distorted in the apoenzyme (Sundstrom et al., 1992).

The GDGX[25-28]NN binding motif is primarily concerned with binding the PP end of the TPP molecule through interactions with a divalent cation. It has been found in TK that the side chains of the aspartate (D) and second glycine residue (G) are important for binding the cation, which in turn forms a complex with the a and P phosphate oxygens of the TPP

molecule (Lindqvist et al., 1992). Although the residues do not directly interact with the TPP molecule any impairment of the metal ion ligands could alter the orientation of the TPP molecule and thus affect the juxtaposition of the catalytic groups (Jordan et a l, 1996). In the POX enzyme the PP group of TPP was found to bind with the metal ion in an octahedral arrangement to Asp447 (D), Asn474 (N) and Gin476 along with residue Gly448 (G) which also binds a phosphate group (Muller and Schultz, 1993). The binding site of TPP in the

9

PDC enzyme from Z mobilis was examined by carrying out site-directed mutagenesis on conserved residues: Asp440 (D), Asn467 (N), Glu449 and Pro459 (Candy & Ronald, 1994). Replacement of Asp440 with Gly, Thr or Asn had previously been shown to result in a completely inactive enzyme with no bound TPP (Diefenbach et al., 1992) however replacement with a Glu residue gave an active enzyme with a much reduced affinity for the cofactors. This indicated that the negative charge is important for cofactor binding which is consistent with the finding that this residue directly binds the Mg2+ atom, in the yeast enzyme (Dyda et a l, 1993). Replacing the conserved Asn467 residue with an Asp resulted in an active enzyme with normal kinetics for pyruvate and Mg2+ but reduced TPP affinity whilst Asn467Gln caused a total loss of TPP binding and activity. This implied that the size of amino acid side chains is crucial in the TPP-binding site, since the additional carbon in a Gin residue would push it further into the coenzyme-binding region. Substitution of the Glu449 residue with an Asp did not affect activity and replacement of the Pro459 with Gly or Ala reduced enzyme stability but not activity.

Whilst the PP end of the cofactor is bound through the co-ordination of the divalent cation to residues in the TPP-binding motif, the thiazolium ring is held in a hydrophobic pocket, at the opposite end of the monomer in TK (Lindqvist et al., 1992). Residues important in the binding of the thiazolium ring are mainly hydrophobic and include, His69 and Gly116; in addition Asp382, whose O^2 oxygen is involved in neutralising the positive charge of the ring and is positioned 3.6A from the C-4' of the thiazolium ring in TK (Nikkola et al., 1994). In PDH-E1 it has been found that the thiazolium ring is probably bound in a hydrophobic region of the (3 subunit (Robinson and Chun, 1993).

What emerges from these studies and comparisons between the TPP-dependent enzymes, is that in all cases TPP is bound in a cleft between subunits. The PP group is bound at the carboxyl end of the strands in the p-sheet of one domain, the thiazolium ring is

located between two domains and the pyrimidine moiety is bound in a hydrophobic pocket consisting mainly of aromatic residues from a different domain in the second subunit. This mode of binding makes the dimer the minimal functional unit (Lindqvist et al., 1992; Muller et al., 1993; Nilsson et al., 1993).

1.5 Biosynthesis1.5.1 Elucidation of the biosynthetic pathway

Thiamine can be synthesised in microorganisms and plants from two precursors - hydroxyethyl thiazole (HET) and hydroxymethyl pyrimidine (HMP). Their phosphorylated counterparts hydroxyethyl thiazole phosphate (HET-P) and phosphomethyl pyrimidine phosphate (HMP-PP) are condensed together to form thiamine mono-phosphate (TMP). In yeast TMP is then dephosphorylated to give free thiamine and subsequently pyrophosphorylated to form TPP, the active form of the vitamin and the end-product of the

10

pathway (Figure 1.5). This process was elucidated from a number of sources. In early experiments it was shown that a thiamine auxotroph of the mould Phycornyces blakesleeanus, was able to grow in the absence of thiamine if 2-methyl-4-amino-5- hydroxymethyl pyrimidine (HMP) and 4-methyl-5-(3-hydroxyethyl thiazole (HET) were

added to the medium, indicating that the biosynthesis of thiamine would involve the independent formation of the two ring structures and their subsequent condensation (reviewed by Leder, 1975). Further, it was shown that HET-P is the sole product of enzymatic activation of the thiazole moiety in yeast (Suzuoki & Kobata, 1960). HMP-PP is formed by two successive phosphorylations, each catalysed by separate enzymes (1) HMP kinase, and (2) phosphomethyl pyrimidine (HMP-P) kinase:(1) HMP + ATP —» HMP-P(2) HMP-P + ATP —» HMP-PP in the presence of Mg2+

HMP-P is therefore an intermediate in this process (Lewin & Brown, 1961).It was subsequently found that thiamine monophosphate (TMP), rather than free

thiamine, is the initial product of the condensation of the thiazole and pyrimidine precursors (Camiener & Brown, 1960; Nose et al., 1961). TMP does not function as a coenzyme and must be converted to TPP. Indeed it had been found as early as 1939 that in yeast TMP is not a direct precursor of TPP (discussed in Hohmann and Meacock, 1998). The detection of the enzyme thiamine pyrophosphokinase (TPK) in yeast led to the view that free thiamine must be an obligate intermediate between TMP and TPP. Subsequently, in 1959 the TPK enzyme was isolated and shown to be inactive towards TMP (Kaziro, 1959). More recently, evidence has been found that HMP-P and HET-P are probably synthesised directly in microorganisms and plants, rather than via their unphosphorylated counterparts, and that HMP and HET are only involved in a salvage pathway from breakdown products or when they are present in the medium - see sections 1.5.2 and 1.5.3.

1.5.2 Origins of the precursors1.5.2.1. Hydroxyethyl thiazole

In E. coli and plants HET appears to be synthesised from cysteine, tyrosine and 1- deoxy-D-r/zreo-2-pentulose (1-deoxy-D-xylulose) (Figure 1.6, panel A). Precursor labelling revealed that the sulphur atom was derived from cysteine (Tazuya et al., 1987) while other studies have shown that the C-2 and N-3 are derived as a single unit from tyrosine (Estramareix & Therisod. 1972). In order to identify the origin of the C-4' C-4 C-5 C-6 C-7 5-carbon unit, l-deoxy-D-r/zreo-2-pentulose and 1 -deoxy-D-em/iro-2-pentulose were used in labelling experiments and it was found that in E. coli the former is more likely to be the direct precursor (Therisod et al., 1981). The l-deoxy-D-r/zrec>-2-pentulose itself has been found to be derived from the condensation of pyruvate and a triose phosphate (White & Spenser, 1982), probably D-glyceraldehyde. Interestingly, TPP-dependent PDH has been shown to catalyse the condensation of pyruvate and D-glyceraldehyde, implicating TPP in

11

Figure 1.5

The thiamine biosynthetic pathway in S. cerevisiae.

The enzymes catalysing the reactions are as indicated:HET-K, denotes hydroxyethyl thiazole kinase; HMP-K, denotes hydroxy methyl pyrimidine kinase; TMP-PPase, denotes thiamine monophosphate pyrophosphorylase; TPK, denotes thiamine pyrophosphokinase; Tr-APase, denotes thiamine-repressible acid phosphatase.The enzymes carrying out steps 1 and 3 are unknown. Step 6 is probably carried out by an unidentified phosphatase.

HETexternal

2-pcntu losc-5-phosplia lc + g lycine + cysteine

histidine + pyridoxine

(from g lu tam ine and 5 phosphoribosy l 1 am ine)

^ ^ i n t e r n a l

2 | h e T-K

HET-P

TMP-PPase

HMP-PP

HMP-K I 4

^internalt

tTransporter

^^^external

TMP 5 7Thiamininternaj ► TPP

TPK

Transporter

Thituriinexternai

§ J T-rAPase

TMP/TPPexternal

Figure 1.6

Biosynthesis of the HET-P precursor of thiamine.

Schematic drawings of the proposed biosynthetic route of hydroxyethyl thiazole phosphate from its precursors:A) in bacteria, from pyruvate, triose, tyrosine and cysteine (adapted from Begley, 1996);B) in yeast, from glucose, glycine and cysteine (adapted from Estramareix and David, 1996).C) The two alternative routes from glucose-6 -phosphate to 2-pentulose-5-phosphate.

A)Pyruvate + triose phosphate

(e.g. glyceraldehyde-3-phosphate)

I

C H 3I

COI

CH*)HI

CH*)H

CH 2O P O 3 H2

1 -deoxy-D-xy lulose- 5-phosphate

CH

2 CH

Cysteine OHCH

Tyrosine

Hydroxyethyl thiazole phosphate

B)

CHO

-O HH O -

-O H—OH

CH2OH

Glucose

CH2OH

COI

CHOHI

CHOH

CH20PO3H2

2-pentulose-5-phosphate

CH

C H ,

-N

, C H 11 Z_

4'A7m—

c h 2o p o 3h 2

Hydroxyethyl thiazole phosphate

Glycine

CO7H

iC H jN H j!

Cysteine

C )D-glucose-6-P — — — — ► D-ribulose-5-P

Oxidative pentose phosphate pathway

Non-oxidative pentose phosphate pathway

D-xylulose-5-P

the synthesis of its thiazole precursor; this phenomenom has also seen in yeast (see next paragraph). That TPP is implicated in its own synthesis is surprising but not unique; the vitamins biotin and pyridoxine are also implicated in their own syntheses (reviewed by Spenser & White, 1997). Recently, it was shown directly by 13C NMR, that the intact C5

chain from l-deoxy-D-r/zreo-2-pentulose is incorporated into the thiazole moiety of thiamine and pyridoxol concurrently, illustrating a link between the biosynthetic pathways of thiamine and pyridoxine (Himmeldirk et al., 1996).

In plants thiazole is formed in the chloroplast from l-deoxy-D-r/zreo-2-pentulose, tyrosine and cysteine, identical with the pathway in E. coli (Juilliard, 1992; Juilliard & Douce, 1991). Using stroma from spinach and maize cell cultures it was again found that 1- deoxy-D-f/zreo2-pentulose was a precursor of vitamin B6 and that this was involved in the synthesis of the thiazole moiety of thiamine. Competition for the common precursor, 1- deoxy-D-r/ireo-2-pentulose, between the biosynthetic pathways of the two vitamins (B1 and B6 ), was proposed and indirect evidence for it was found when cells growth-arrested in thiamine free-medium were restored to growth by the addition of thiazole to the medium or the omission of pyridoxine (Juilliard, 1992). The pentulose involved in HET formation in bacteria and plants is likely to be l-deoxy-D-xylulose-5-phosphate (reviewed by Begley, 1996); if this is the case, then HET-P is probably formed directly rather than via the unphosphorylated intermediate.

In yeast and aerobic bacteria (such as Bacillus subtilis and Pseudomonas putida) the thiazole precursor is formed via a different route to that found in enteric bacteria and plants (Figure 1.6, panel B). In S. cerevisiae it was found that the methylene carbon atom of glycine, not tyrosine, was the origin of the C-2 N-3 of thiazole (White & Spenser, 1979). However the sulphur atom is thought to be derived from cysteine, as in enteric bacteria and plants, contrary to previous reports which suggested that the 5-methyl group of methionine was the source. In 1982 it was already known that the other precursor for HET was likely to be a 2-pentulose-5-phosphate, therefore experiments were carried out in order to determine which 2-pentulose-5-phosphate molecules might be the source. Although yeast cells are unable to take up pentoses they are produced from hexoses by the pentose phosphate pathway, therefore 14C glucose was used in labelling studies. It was found that either D- ribulose-5-phosphate or D-xylulose-5-phosphate (products of D-glucose-6 -phosphate by the oxidative and non-oxidative pentose phosphate pathways, respectively) could act as a precursor of the thiazole C-5 chain, the contribution made from each pathway depended on the growth development of the micro-organism and the carbon source being used (White and Spenser, 1982). The non-oxidative route is catalysed by the TPP-dependent enzyme, transketolase. This may explain why a second route via the oxidative pathway, which does not rely on thiamine itself, exists. If D-ribulose-5-phosphate or D-xylulose-5-phosphate are substrates for the thiazole moiety of thiamine then again it would reveal HET-P as the most probable direct product, rather than the unphosphorylated HET.

12

1.5.2.2 Hydroxymethyl pyrimidineAlthough HMP is structurally similar to the pyrimidines of nucleic acids, it was

found not to share a common biosynthetic pathway with them. It became clear that instead the pyrimidine molecule shared a common pathway with purine bases when evidence was found that glycine was a precursor for both pathways, in bacteria. Early work revealed single-site mutations in S. typhimurium and E. coli which led to simultaneous purine and thiamine auxotrophies (reviewed by Young, 1986). Subsequently, in S. typhimurium it was found that 5-aminoimidazole ribonucleotide (AIR), an intermediate in the formation of purine nucleotides, was also an intermediate in the formation of thiamine and marked the branch point for the two pathways (reviewed by Leder, 1975). Evidence for two alternative pathways for the synthesis of pyrimidine has recently been discovered in S. typhimurium (reviewed by Begley, 1996). These also use AIR as an intermediate, but they are independent of the purine pathway. In E. coli, the pyrimidine precursor was similarly found to be derived from AIR.

The first step in HMP biosynthesis from AIR involves the cleavage of the bond between C-3' and C-4' of the ribose moiety, followed by the addition of a two carbon fragment. Using radiolabelled 14C compounds it was found that this two-carbon unit was also derived from AIR, being the C-4' and C-5' of the ribose moiety (Yamada & Kumaoka,1983). The two carbons form a ring between the C-4' and C-5' of the imidazole, which then opens out to form the pyrimidine ring (Figure 1.7, panel A). Thus, in bacteria the pyrimidine subunit is derived solely by rearrangement of AIR. It was proposed that the product formed from this reaction was HMP-P, rather than HMP, since if HMP were synthesised via this route then 5-aminoimidazole riboside would be the precursor rather than AIR. This would mean that HMP was not an obligate intermediate in thiamine biosynthesis. The phosphorylated precursors are probably involved in the biosynthetic pathway, whilst the unphosphorylated products form part of a salvage pathway following degradation of thiamine and phosphorylated intermediates.

As is the case for HET, the route for synthesis of HMP differs in yeast to that found in E. coli and S. typhimurium . Although AIR is a precursor for the purine bases of nucleic acids in yeast, it is not thought to be a precursor for thiamine biosynthesis. Studies suggest that in S. cerevisiae, HMP is derived from the imidazole ring of histidine and ribose. Incorporation studies revealed that the C-6 , C-5 and C-5' of pyrimidine is derived from the C-l, C-2 and C-3 atoms of ribose (Tazuya et a l, 1986). Later these particular atoms were found to be incorporated into the pyrimidine ring, via the C-6 , C-5 and C-5' of pyridoxine (vitamin B6 ). In addition it was shown recently that the C-2, C-2' and N-l atoms of HMP are also derived from pyridoxine, indicating that the vitamins, B 1 and B6 , share a common biosynthetic pathway (Tazuya et al., 1994). The N-l atom of pyridoxine had already been shown to be derived from glutamine, thus it was postulated that glutamine and ribose may be incorporated into the N -l, C-6 , C-5, and C-5' atoms of pyridoxine as 5-phosphoribosyl-l-

13

Figure 1.7

Biosynthesis of the HMP-P precursor of thiamine.

Schematic drawings of the proposed biosynthetic route of hydroxymethyl pyrimidine phosphate from its precursors:A) in bacteria, from AIR (taken from Yamada and Kumaoka, 1983);B) in yeast, from histidine and pyridoxine (adapted from Tazuya et al., 1995).

A) 4 r 1>s F j j

H2N N f

N3

2 O3POH2C

HO OH

5’2-O3POCH2

H dC H

HOCH HCI

OH

N32

H2N Nt Ij)

CHOH

2-03P0CH2CHjj |J H 2 N ^ N 7

N3

2

CHOHICHOHICHO

n h 2

HMP-P

B)

HO ^OHH20 3P0H2C^CHiCH OH

I \ /CHCHOH\

NH2

5-PHOSPHORIBOSYL- 1-AMINE

HMPr hoh2c

ch2oh1-------------------- \

HOH2C s4L_ _ _

PYRIDOXINE

HISTIDINE

GLUTAMINEAMIDE-N

amine (a precursor of AIR); these atoms would then be incorporated into HMP (Tazuya et al., 1995). Other studies with 15N labelled histidine revealed that the N-l and N-3 atoms of histidine are incorporated into the pyrimidine ring and it was suggested that the N-3, C-4 and amino-N of pyrimidine were derived as a single unit (Tazuya et al., 1989). Thus HMP is derived from histidine and pyridoxine molecules (Figure 1.7, panel B). However it is not yet clear whether the immediate precursor of pyrimidine is pyridoxine itself or a derivative of it such as, pyridoxal or pyridoxal phosphate, in which case HMP-P or HMP-PP would most likely be the product, rather than HMP, again indicating that the unphosphorylated precursor is not an obligate intermediate in the biosynthesis of thiamine.

There is new evidence that there are two pathways leading to HMP biosynthesis in yeast. In the minor pathway formate provides the C-2 atom while the C-5 and C-4 atoms of pyrimidine come from the C-1 and C-2 carbons of a pentulose. However the source of the C-2' C-5' and C- 6 atoms is unknown (reviewed by Begley, 1996).

1.5.3 Genetics of thiamine biosynthesis in bacteriaIn E. coli: five kinases involved in TPP biosynthesis have been identified. These

include a thiamine kinase, encoded by thiK and, a thiamine monophosphate kinase, encoded by thiL; the latter enzyme was found to be essential. Therefore in E. coli the phosphorylation of thiamine appears to occur via two separate reactions rather than by a pyrophosphorylation, as in yeast. The other E. coli kinases include: a HMP-P kinase, encoded by thiD\ a HMP kinase, encoded by thiN\ and a HET kinase, encoded by thiM; the thiM and thiK products are thought to be salvage enzymes as they have been shown to be non-essential. This genetic evidence reinforces the biochemical data which suggest that the phosphorylation of the precursors is not an essential part of thiamine biosynthesis. Whether ThiNp is essential has not yet been determined (reviewed by Begley, 1996).

Five other thiamine biosynthetic genes have been identified in E. coli, located in a cluster at 90 minutes - thiCEFGH. These genes are tightly linked and coordinately expressed (Kawasaki & Nose, 1969) but appear to have discrete promoters (Vanderhom et al., 1993). In addition to these and the kinase genes, two other E. coli genes are known - thiJ and thiB. Mutations in thiF, thiG and thiH were found to be defective in HET synthesis; these did not respond to the precursor of the thiazole moiety, l-deoxy-D-f/zreo-2-pentulose, and were therefore thought to be blocked in steps leading to its conversion to HET. The product of thiF may play a role in the transfer of the sulphur atom to the thiazole moiety but the exact roles of thiG and thiH are unknown. The thiJ gene product is also involved in HET synthesis, but again its exact function is not known. The thiEp product, along with thiBp, is responsible for the coupling reaction of HET-P and HMP-PP. Only the thiC gene has been implicated in the synthesis of HMP, in E. coli (reviewed by: Begley, 1996; Spenser and White, 1997).

14

1.5.4 Genetics of thiamine biosynthesis in yeastThe genetics of thiamine biosynthesis in yeast is less well understood. Only three

biosynthetic genes and four thiamine auxotrophic strains had been identified in S. cerevisiae at the start of this study. The first thiamine mutant, isolated in 1960, was designated thil (Hawthorne & Mortimer, 1960). Other than its thiamine requiring phenotype and its assignment to the right arm of chromosome XIII, no further characterisation of this strain had been published. Two other thiamine auxotrophic mutants had been isolated from the budding yeast, thi2 and thi3, which were thought to be defective in positive regulatory factors (Kawasaki et al., 1990; Nishimura et al., 1992b). The other auxotrophic mutation, thi6, was thought to be a structural gene in thiamine biosynthesis, encoding a bifunctional enzyme with HET-kinase and TMP-PPase activities (Kawasaki, 1993). One of the three known thiamine biosynthetic genes, THI80, was found to encode the structural enzyme for TPK (Figure 1.8, step 7). The other two genes were shown to be involved in precursor biosynthesis; THI4, implicated in HET-P biosynthesis and THI5, in HMP-PP biosynthesis (Figure 1.8 steps 1 and 3, respectively). The exact functions of the products of these latter two genes are still unknown.

The isolation and characterisation of all of these genes and mutations is discussed below, apart from thil the analysis of which is described fully in Chapter 5.

1.5.4.1 T H I 4The THI4 cDNA (originally denoted MOL1) was isolated in this laboratory during a

differential screen of exponential and near-stationary phase cells, as a result of its strong induction when a culture grown in molasses medium moved into stationary phase (Praekelt & Meacock, 1992). Later, it was found that the repressing component was the water soluble vitamin B l, thiamine. Studies on THI4 expression, using a promoter-/«cZ construct based on a centromeric plasmid, showed that in the absence of thiamine high levels of p-

galactosidase activity were obtained. That the TH14 gene is highly transcribed is reflected in the observation, through SDS-PAGE, that Thi4p is probably one of the most abundantly synthesised proteins under non-repressing conditions, possibly indicating a need for it to compete with other proteins in different pathways for common substrates.

The presence of an adenine dinucleotide binding site within the deduced amino acid sequence gave the first clue as to what the function of the Thi4p protein might be - a biosynthetic protein with possibly oxidase or dehydrogenase activity (Praekelt and Meacock, 1992). Later it was found that a thi4 null mutant was a thiamine auxotroph which was restored to growth by supplementation with the HET precursor, thus indicating a role in the synthesis of the thiazole moiety of the vitamin (Praekelt et al., 1994). Subsequently the homologous gene nmt2 (thi2) was isolated from fission yeast and this was also shown to function in HET biosynthesis (Manetti et al., 1994; Zurlinden & Schweingruber, 1992).

15

Figure 1.8

Genes involved in the thiamine biosynthetic pathway of S. cerevisiae.

Genes involved in the pathway leading to the formation of TPP are as indicated.

external

2-pen tu losc-5 -phos i 1 . ‘e + g lycine + cysteine

histidine + pyridoxine

(from g lu tam ine and 5 -p h o sp h o r ib o sy l-1 -amine)

THI4

THI5 THU 1 THI12 THI13

HFT-noA internal

2 | THI6

IIET-P

TH16

HMP-PPt^^^internal

I THI10

HM PCxtcrnal

6T M P ► T h ia m in internal

THI80t THI10

Thi;uiiincxtcn)a|

8 T P H 0 3

T M P /T P P exlernal

T P P

44

The deduced amino acid sequence of Thi4p is similar to stress proteins of two Fusarium species: 59.2% identity with STI35 of F. oxysporum and 57.7% identity with STB5 of F. solani (Choi et a l, 1990; Praekelt et a l, 1994), although how Thi4p might function in stress response is obscure. A number of other homologues of the ScTHI4 gene have now been found in various plant species. Surprisingly these were not isolated directly as thiamine genes but as result of screens for genes involved in developmental processes. The thil gene of maize (Zea mays) was found to be induced in early embryo development (Belanger et al., 1995) and the homologue in Alnus glutinosa, also termed thil, was found during an investigation into nodule development after Rhyzobium infection (Ribeiro et a l,1996). A requirement for increased thiamine biosynthesis in these highly metabolising cells might be expected. However, perhaps the most surprising was the isolation of the Arabidopsis thaliana homologue by its ability to complement DNA damage repair mutants of E. coli (Machado etal., 1996).

A mutant E. coli strain carried defects in the initial steps of base excision repair and was hypersensitive to agents producing oxygen free-radicals and to alkylating agents. The thil gene product was directly implicated in the repair of damaged DNA, rather than in the prevention of the DNA lesions, as a uvrA mutant expressing Thilp was able to reactivate introduced UV damaged X phage DNA. Expression of the th il cDNA in S. cerevisiae

complemented a thi4::URA3 mutant to thiamine prototrophy confirming a role in biosynthesis. In addition, the S. cerevisiae THI4 gene was able to partially correct the E. coli DNA repair deficiency, hence a possible role for the gene in repair of DNA damage of yeast cells was investigated. Wild-type (THI4) and thi4::URA3 strains were found to be equally sensitive to UV and MMS treatment but colonies from the thi4 strain were smaller than wild- type and showed a petite phenotype i.e. no growth on non-fermentable carbon sources. The addition of thiamine or thiazole to the growth medium did not reduce the incidences of respiratory mutants arising in the thi4 strain suggesting that it may be the Thi4p itself which is important in the repair and not its function in HET synthesis (Machado et al., 1997). Although in S. cerevisiae expression of the TH14 gene is repressed by thiamine, a residual low level expression was measured; this must be sufficient for DNA damage tolerance, as increased TH14 expression did not increase tolerance.

It has been suggested that both S cTH14 and At thil contain DNA binding motifs and sequences homologous to bacterial DNA polymerases (Machado et al., 1997) which could be implicated in DNA damage tolerance. Additionally the dinucleotide binding domain found in Sc THI4 indicates an enzymatic activity and possible RNA binding properties for the protein. However the enzyme may well have just one activity which acts on different substrates - for thiamine biosynthesis and for modifying nucleic acids. If this is the case it would not be the only example of a biosynthetic gene that has a dual role in nucleic acid metabolism: the E. coli gene nuvC (thiJ) is thought to have a dual role in thiazole biosynthesis and modification of uridine to 4-thiouridine in tRNA (Ryals et a l, 1982); the

16

yeast ILV5 gene, involved in branched-chain amino-acid biosynthesis, was also found to be involved in mtDNA stability (Zelenaya-Troitskaya et al., 1995).

The Thi4p is thought to be targetted to the mitochondria, as it has an N-terminal region characteristic of a mitochondrial transit peptide. The Alnus homolog was found to complement the thi4::URA3 strain only when the N-terminal yeast Thi4p leader sequence was added to the 5' end of the cDNA and indeed the other plant homologues were able to rescue the yeast mutant more efficiently when similarly modified. Thus it was concluded that the S cTHI4 and At thil gene have dual functions in thiamine biosynthesis and mtDNA damage tolerance and that THI4 plays a role in maintenance of biological activity of mtDNA (Machado et a l, 1997).

1.5.4.2 THI5The TH15 cDNA was isolated, in this laboratory, at the same time as THI4 cDNA. It

is homologous to the thi3 (nm tl) gene of the fission yeast, showing 62% identity at the amino acid level (Hather, 1996). This S. pombe gene was isolated as a result of a differential screen between cultures grown in the presence and absence of thiamine and was the first fully regulatable gene isolated from this yeast (Maundrell, 1990). A very high level of the message is transcribed in the absence of thiamine and it is totally repressed in 2jiM of

thiamine, hence nmtl - no message in fhiamine. The nmtl protein was shown to be involved in the the synthesis of the pyrimidine moiety of thiamine. Thus it was assumed that THI5 is involved in this part of the pathway in the budding yeast.

A homologue of THIS has also been isolated from the dairy yeast K. lactis (Walsh and Meacock, unpublished results). Although it was found that there is only one copy of this gene in the S. pombe and K. lactis genomes, four copies of the THI5 sequence have been identified in the S. cerevisiae genome (Hather, 1996). The four genes are designated: THI5 (YFL058w), located on chromosome VI; THI11 (YJR156c), located on chromosome X; THI12 (YNL332w), located on chromosome XIV; and THI13 (YDL244w), located on chromosome IV. The proteins show around 99% amino acid identity. Of the 27 changes in the nucleotide sequences 24 are in the third base position resulting in only 3 amino acid differences in the gene products. Divergence between the four sequences begins approximately 700bp upstream and approximately 300bp downstream of the coding sequence. The TH15 and THI12 genes are located within the left subtelomeric region of their respective chromosomes and are more similar to each other than to either THI11 or TH113', likewise, THI11 and THU3 are more similar to each other than to THIS or THI12 and are located within the sub-telomeric region of their respective chromosomes. The TH15 and THI12 loci have been shown to be active since their promoters fused to the E. coli LacZ reporter gene give (3-galactosidase activity, in the absence of thiamine (Burrows, 1997).

Whether the other two loci are actively transcribed is yet to be tested, hence THI11 and TH ll3 may be redundant.

17

Thus the THIS gene appears to have been duplicated between four different chromosomes, perhaps with the duplications to TH15 and THI12, and, THI11 and THI13 being most recent. It is predicted that either the duplications have occurred recently or a high selective pressure has maintained conservation of the genes, this last option being most likely as each gene is highly conserved whilst the flanking DNA is less so. Why there should be four copies of this gene, with a presumed common function in biosynthesis of the pyrimidine precursor of thiamine is perplexing. One explanation could be that as the THI4 gene is expressed to a level approximately four times higher than either the THI5 or THI12 genes, ascertained by p-galactosidase assays (Burrows, 1997), then expression from four

THIS-like genes may maintain a balance in the levels of each precursor in the flux through the pathway.

1.5.4.3 T H I 6A strain carrying the thi6 mutation was isolated on the basis of its resistance to 2-

amino-HET, an inhibitor of thiamine biosynthesis in microorganisms, which specifically targets the HET kinase and TMP-PPase enzymes (Kawasaki, 1993). The resistant mutant was shown to have reduced activities of HET-kinase and TMP-PPase, but not HMP-kinase or HMP-P kinase. Genetic analysis revealed that the mutation was recessive, in a single gene and that the phenotypes of the reduced enzyme activities cosegregated with the phenotype for the resistance to 2-amino-HET. Additionally, the two activities of HET-kinase and TMP- PPase (Figure 1.8 steps 2 and 5, respectively) were copurified to apparent homogeneity. These studies imply that both activities are contained on a single bifunctional enzyme encoded by the THI6 gene. Subsequently, during the course of this study, the THI6 gene was cloned following complementation of a thi6 mutant strain. This was possible since a wild-type yeast cell is able to grow on the thiamine antagonists, pyrithiamine and oxythiamine, in the absence of the vitamin, as thiamine can be synthesised from the pyrimidine moiety of pyrithiamine and the thiazole moiety of oxythiamine. The HET kinase deficiency of the thi6 strain, however, renders this strain incapable of growth on the two antagonists; thus THI6 was isolated by suppressing this growth defect. Characterisation of this gene is discussed further in Chapter 4.

A homologue of ScTHI6 has been isolated from 5. pombe. This gene, thi4, encodes a 518 amino acid protein with a predicted 55.6 molecular mass. It is also thought to be bifunctional with TMP-PPase and HET kinase activities (Zurlinden & Schweingruber, 1994). The amino acid sequence of thi4p is homologous with the thiE protein of E. coli, which is responsible, along with thiBp, for the coupling reaction of HMP-PP and HET-P.

1.5.4.4 T HI 8 0The thi80-l mutation was isolated as a transport mutant, following EMS mutagenesis

(Nishimura et a l, 1991) using a screen based on the staining method described by Iwashima

18

et al. (1981). Triphenyltetrazolium chloride (TTC), a basic dye, is taken up by yeast cells via the thiamine transport system when the cells are grown in thiamine-free medium and is reduced to give a red coloration. However when the pathway is repressed by exogenous thiamine the cells do not take up the dye and remain cream coloured. Thus, a mutant with constitutive thiamine transport activity which turned red in the presence of thiamine and TTC, was identified. Subsequent analysis showed this mutation, designated thi80-l, to be recessive, in a single gene and responsible for high resistance to oxythiamine, a thiamine antagonist with a potency dependent on TPK activity, suggesting that the strain had a defect in this enzyme. Enzyme assays confirmed that the thi80-l mutant had reduced TPK activity, the enzyme responsible for catalysis of thiamine to TPP (Figure 1.8, step 7). As a result of this defect the mutant contained about half the intracellular TPP level of wild-type cells when grown with 0.2pM exogenous thiamine. The mutant also displayed constitutive activities of

thiamine transport, T-rAPase and the enzymes involved in synthesis of thiamine from HET and HMP. This phenotype of constitutive enzyme activities indicated that it is TPP which acts as a negative effector, in the utilization and synthesis of thiamine in S. cerevisiae rather than thiamine itself.

The TH180 wild-type gene was isolated on the basis of its ability to complement the constitutive T-rAPase activity of the thi80-l mutant strain, again using the staining method as a screen (Nosaka et al., 1993). Heterologous expression of the ORF in an E. coli mutant strain lacking TPK activity resulted in detection of marked TPK activity, confirming that THI80 encodes the TPK structural enzyme. A gene disruption at the THI80 locus was lethal, revealing that Thi80p is essential and is the only enzyme able to catalyse the conversion of thiamine to TPP, in yeast.

1.6 The thiamine transport pathwayAs well as synthesising thiamine de novo, microorganisms are able to take up the

vitamin efficiently from the external environment. The transport process in yeast and E. coli display characteristics of a carrier mediated active process. In S. cerevisiae thiamine uptake is energy (glucose), temperature and pH dependent (optimum at pH 4.5) and acts against a concentration gradient 10,000 times greater than the external concentration. Metabolic inhibitors such as fluoride ions, iodoacetate and nitrite ions were shown to inhibit thiamine uptake in baker's yeast, confirming that thiamine transport is energy dependent (Iwashima et al., 1973). It is also known that uptake is growth phase dependent, being optimal when cultures are in log-phase (Iwashima et al., 1979). Structural specificity of the system has been demonstrated; the pyrimidine moiety appears to be recognised since analogues such as oxythiamine (which has a modified pyrimidine moiety) did not compete to inhibit transport of 14C thiamine, whereas analogues with a modified thiazole moiety but a normal pyrimidine

19

subunit (such as pyrithiamine, chloroethyl thiamine and dimethialium) did inhibit uptake of 14C thiamine.

In E. coli thiamine is phosphorylated immediately, possibly as a means of metabolic trapping, since phosphorylation deficient mutants exhibit counterflow efflux of the vitamin (Kawasaki and Nose, 1969). It was first thought that yeast cells, in contrast to E. coli, retained thiamine in the free form (Iwashima et a l, 1973). However other groups have since found that after a short time, most of the vitamin in S. cerevisiae is in fact converted into the pyrophosphate form. Indeed it was shown that approximately 60% of the intracellular thiamine was in the form of TPP, in wild-type cells grown in 0.2|iM thiamine (Nishimura et

a l, 1991; Ruml e ta l , 1988).The transport system is irreversible in S. cerevisiae illustrating a further difference

from the situation in the prokaryotic system. No excretion or counterflow efflux of thiamine was seen either in a wild-type strain or in a strain with lowered thiamine phosphorylation activity (Ruml et a l, 1988). Indeed oxythiamine, an inhibitor of thiamine phosphorylation, did not induce release of thiamine from the cell, but it did lower the rate of uptake; further when TPK activity was reduced, in a thi80-l mutant, then most of the intracellular thiamine was present in the unphosphorylated form but this was not lost from the cell (Nishimura et a l, 1991; Nosaka er <z/., 1993).

1.6.1 Proteins involved in transportBiochemical studies revealed the presence of two thiamine binding proteins in S.

cerevisiae, which were localised to the periplasmic space and the plasma membrane (Iwashima & Nishimura, 1979; Nishimura et a l. 1986). The soluble periplasmic thiamine binding glycoprotein was isolated after growth of cells in thiamine-free medium and cold osmotic shock treatment. The protein was 140Kd in size, was regulated by exogenous thiamine and specifically bound thiamine with a K<} of 29nM, suggesting that it is involved

in the transport process. However this dissociation constant for thiamine-binding is about six fold lower than the apparent Km (180nM) of thiamine transport and since structural

specificity of this protein was not always reflected in transport specificity, this protein is probably not responsible directly for the transport of thiamine. Furthermore, a thiamine transport mutant was found to contain as much soluble thiamine-binding protein as wild- type. Thus another protein must be involved. The membrane-bound protein was found to have an apparent Kd of 170nM, an optimal pH of 5.0 for binding and structural specificity similar to the transport pathway. Additionally, it was shown that the thiamine binding activity of the membrane fraction in the transport mutant was 3% of wild-type; therefore it was concluded that this protein has a direct role in thiamine transport in S. cerevisiae (Iwashima et a l, 1979).

The 140Kd soluble periplasmic protein was later identified as a thiamine-repressible APase, which was required for hydrolysis of thiamine phosphates before uptake into yeast

20

cells. The protein had been shown to have high affinity for thiamine phosphates with a Km value in the micromolar range and mutations affecting this enzyme resulted in strains that were impaired for TMP and TPP uptake but not for uptake of free thiamine. Also it was found that thiamine competitively inhibited the thiamine monophosphatase activity of the T- rAPase (Nosaka, 1990; Nosaka et al., 1989).

1.6.2 Genes involved in thiamine transportEarly work revealed the presence of two kinds of acid phosphatase enzymes in S.

cerevisiae (Toh-E et al., 1975b; Toh-E et al., 1973). One of these appeared to be constitutive and was encoded by phoC, later renamed PH03; the enzyme was subsequently found to be repressible by thiamine (T-rAPase). The other phosphatase was repressible by inorganic phosphate (Pi-rAPase) and was encoded by phoE, later renamed P H 05 (Bajwa et al.,1984). Then a pho3 mutant was isolated which was unable to take up thiamine in the form of its monophosphate or diphosphate ester, indicating that the Pi-rAPase was ineffective towards phosphorylated thiamine substrates (Nosaka et al., 1989). Therefore it was concluded that PH03 encoded the T-rAPase.

The T-rAPase carries out the dephosphorylation of thiamine phosphates found in the external environment in the periplasmic space, but it is not directly involved in the transport of thiamine per se; (an)other protein(s) must therefore be involved, possibly the membrane bound thiamine binding protein identified by (Iwashima et a l, 1979). Recently, a gene encoding a thiamine transporter protein was isolated and characterised by two different groups: TH17, isolated by (Singleton, 1997) and TH110 (Enjo et al., 1997). In both cases the gene was isolated on the basis of pyrithiamine resistance. When pyrithiamine is taken up by the thiamine transport system it inhibits the essential enzyme TPK and thus is lethal to yeast cells (Iwashima et al., 1975). Therefore mutants with reduced thiamine transport are more resistant than wild-type.

In the first case a pyrithiamine resistant mutant strain was made conditionally defective in thiamine biosynthesis: the thiamine biosynthetic gene TH14 was disrupted and the strain was transformed with a wild-type TH14 gene contained on a plasmid also bearing the URA3 gene. Growth was therefore restricted on medium containing low levels of thiamine (0.12p.M) and 5-fluoroorotic-acid (5-FOA, a chemical which is converted to the

toxic substance 5-fluorouracil through decarboxylation by the product of the URA3 gene), since only ura3 cells that had lost the plasmid would be able to grow. The gene isolated by complementing this growth defect was confirmed to be a thiamine transporter when disruption of the gene led to an inability to take up thiamine. The gene was designated THI7 and is located on chromosome XII.

In the second case, the TTC staining method for identifying thiamine transport mutants (described in Section 1.5.4.4) was used to screen for complementing clones for a pyrithiamine resistant mutant. The gene isolated, TH110, was shown to be a thiamine

21

transporter by the following results: a membrane fraction of a thilO null strain displayed none of the thiamine transport activity or thiamine-binding ability displayed in a wild-type strain; these activities were restored when the TH110 ORF was expressed from a GAL1 promoter; the THI10 gene was found to be homologous with two other yeast transporter genes, DAL4, involved in allantoin transport, and FUR4 involved in uracil transport. Therefore it was concluded that the thilOp (thi7p) is located in the plasma membrane of yeast cells, has thiamine binding ability and is involved in transport. However, whether this protein is identical with the membrane-bound thiamine-binding protein identified by Iwashima et al. (Iwashima et al., 1979) is not known.

A database search of the published sequence of the S. cerevisiae revealed two other unknown ORFs that share homology with THI10 - YOR071c and YOR192c, both of which are situated on chromosome XV (Enjo et al., 1997). These putative genes, when expressed from the GAL1 promoter, showed 13.7% and 41.0% of the activity of the THI10 ORF, in a thilO background. Therefore protein products of these genes may be involved in a thiamine transport complex with thilOp (thi7p). Alternatively, they could be involved in a different transport system which has a weak affinity for thiamine. Another mutation in S. cerevisiae leading to complete loss of thiamine uptake was identified and designated thpl (Ruml & Silhankova, 1996). Since it was mapped to the left arm of chromosome VII it appears to differ from the THI7 / TH110 transporter gene. It is not known whether this mutation represents another structural thiamine transporter or a gene involved in the regulation of thiamine transport.

A single gene has been found in S. pombe responsible for thiamine transport and a mutation in this gene also leads to pyrithiamine resistance (Schweingruber et al., 1991).

1.6.3 Transport of precursorsIt is thought that HET uptake occurs via simple or faciliated diffusion followed by

metabolic trapping, mediated by rapid phosphorylation to HET-P. Uptake of hydroxyethyl [2-14C] thiazole was studied in S. cerevisiae and it was found that after two minutes incubation 80% of radioactivity was in the form of HET-P: no appreciable uptake was observed in a HET kinase mutant (Iwashima et al., 1986). Therefore uptake of this precursor requires the activity of the HET-kinase enzyme.

The HMP precursor, on the other hand, is taken up by the same carrier-mediated active transport system as thiamine. It was shown that tritiated HMP was taken up by resting yeast cells in an energy (glucose), temperature and pH dependent manner. Moreover thiamine is a competitive inhibitor of the process and specific inhibitors of the thiamine transport pathway, pyrithiamine and 4-azido-2-nitrobenzoyl thiamine, inhibited uptake of HMP (Iwashima et al., 1990a). A mutant defective in thiamine transport also showed reduced uptake of HMP. However the presence of other possible HMP transport pathways

can not be excluded, since in contrast to thiamine, HMP uptake displays counterflow efflux whereby the accumulated precursor is released from the cells (Iwashima et al., 1990b).

1.7 Regulation of thiamine metabolismThe mechanisms by which thiamine metabolism is regulated are still not fully

understood, although regulation of thiamine biosynthesis downstream from the prescursors and of the transport pathway has been partially clarified. A number of mutations have been shown to effect regulation in S. cerevisiae, thi2, thi3, thi80-l and a set of det mutations isolated in this laboratory, and models for how these components may interact have been proposed (Burrows, 1997; Nishimura e ta l , 1992b).

1.7.1 The thi2 and thi3 mutationsThe thi2 (pho6) strain was isolated initially through a defect in acid phosphatase

activity (Toh-E etal., 1973). Following EMS mutagenesis, mutants were screened using the diazo-coupling reaction described by (Schurr & Yagil, 1971), where mutated colonies were spread onto medium containing high or low concentrations of inorganic phosphate (according to the mutants being sought) and were overlaid with soft agar containing a-

naphthyl-phosphate and Fast blue salt. An active acid phosphatase activity in the cells would result in a dark red coloration developing within 30 - 60 minutes, whereas lack of activity in the colony would result in it remaining white. Strains displaying reduced APase activity on low levels of Pi were isolated and from these two genes involved in controlling Pho3p, the "constitutive acid phosphatase" (T-rAPase), were identified, PH 06 (phoF) and P H 07 (phoG) (Toh-E et al., 1975a). Mutations in these genes had no effect on the Pi-rAPase. Later it was found that the thi2 strain also had reduced activities of the thiamine biosynthetic enzymes (Kawasaki et a l, 1990). Thus the THI2 gene is involved in the positive regulation of T-rAPase activity and the thiamine biosynthetic enzymes.

The thi3 mutation was isolated following EMS mutagenesis, due to an inability of the strain to grow on thiamine-deficient medium. The treated cells were enriched for thiamine mutations using nystatin and strains which grew on thiamine-containing medium but not on thiamine-free medium were isolated. The thi3 mutation was found to be recessive, in a single gene and led to reduced activities of the T-rAPase and the thiamine biosynthetic enzymes; in addition it resulted in a marked reduction in thiamine transport. These phenotypes all cosegregated with the thiamine auxotrophy; thus the TH13 gene appeared to encode a positive regulatory factor differing from Thi2p in the regulation of thiamine transport (Nishimura et al., 1992b). From this evidence a model was proposed for how thiamine biosynthesis is regulated in S. cerevisiae (Figure 1.9). This hypothesis assumes that the positive regulatory factors, Thi2p and Thi3p, switch on all the genes involved in thiamine metabolism, but the Thi3p alone positively activates the thiamine transport system and that

23

Thi3p Thi2p

r TPP > (EFFECTOR)

ENZYMES PH 03TRANSPORT

Figure 1.9

Model for the interaction between regulatory factors involved TPP biosynthesis.

Square boxes indicate genes; ovals represent regulatory factors; arrows, indicate stimulatory effects; horizontal bars, indicate repressive effects; ?, represents negative regulatory factor(s). Adapted from Nishimura et al. (1992b).

the 777/2 and 77773 genes are negatively regulated by intracellular TPP levels, either directly or indirectly.

Although complementing clones have been isolated for thi2 (Nishimura et al., 1992a) and thi3 (Nishimura et al., 1992b) the wild-type ORFs have not yet been identified.

Studies on the thi80-l mutant strain revealed that the thiamine metabolic pathway is under negative regulation mediated by the level of intracellular TPP (see Section 1.5.4.4). Northern blot analysis revealed that the THI80 gene itself is regulated to some extent by intracellular TPP levels, and it requires the positive activation of THI2 and THI3 (Nosaka et al., 1993). Intracellular TPP levels have now been shown to regulate the expression of all other thiamine biosynthetic genes so far isolated - THI4, 77/75, THI6 and PH03 (Burrows,1997). However, unlike T-rAPase and the genes involved in thiamine biosynthesis, THI80, was found to be expressed constitutively at a low level and was incompletely repressed by exogenous thiamine. This would be expected since cells require TPK activity to convert the transported thiamine to TPP.

1.7.2 Regulation of biosynthesisBecause thiamine biosynthesis is energy expensive to the cell, involving a number

enzymic reactions (including phosphorylation, dephosphorylation and condensation), regulation of uptake against biosynthesis is tightly regulated. Northern blot analysis revealed that upon addition of a repressing concentration of thiamine (2|iM) the TH14 transcript was

undetectable within 20 minutes (Praekelt et al., 1994). Moreover, the minimal intracellular concentration of thiamine required to repress TH14 gene expression in S. cerevisiae was found to be approximately 2 0 pmol/1 0 7 cells - only double the endogenous level of intracellular thiamine (Praekelt et al., 1994), revealing that this highly transcribed gene is tightly regulated and rapidly repressed. Northern blot analysis has also shown that the highly expressed THI5 transcript is completely repressed by the addition of thiamine to the medium, indicating that the regulation of 777/5 like THI4, is tightly controlled (Hather, 1996).

The 777/5 homologue in S. pombe, thi3, also produces a high level of transcript and overexpression of this gene led to a 1 0 % increase in growth rate similar to the enhanced growth rate of wild-type cells in minimal medium supplemented with thiamine (Maundrell, 1990). Thus it was thought that thi3p may carry out a rate-limiting step in thiamine biosynthesis. It would be interesting to see whether growth enhancement occurs with the overexpression of THI5 genes, or indeed 77/74, in S. cerevisiae. That the genes are highly expressed and tightly controlled may suggest that they do carry out rate-limiting steps in thiamine biosynthesis.

In our laboratory a number of regulatory factors have been identified, in S. cerevisiae, through a set of det mutant strains that displayed derepressed expression of a THI4-lacZ reporter gene in medium containing thiamine. Phenotypic analysis revealed that three partially dominant and one recessive det mutant displayed derepressed TH14-lacZ

24

expression levels comparable to a wild-type Det+ strain grown in inducing conditions; none of these mutations were allelic with thi80, nor were they transport mutants. Therefore they must define either new mutations in the positive regulatory genes -TH12 and TH13 - or novel thiamine regulatory genes. These pleiotropic mutations cause derepression of all thiamine- regulated genes tested, suggesting that thiamine genes are controlled by a common set of regulatory factors (Burrows, 1997). Whether these genes are homologous to the tnr mutations in S. pombe is not yet known.

1.7.3 Regulation of the transport pathwayThe uptake system of thiamine in yeast is regulated by thiamine. This is thought to be

mediated through repression rather than feedback inhibition (Iwashima & Nose, 1976). It has been demonstrated that cells grown in excess thiamine took up the vitamin to maximal internal concentration of 1600pmol/107 cells whence the transport system was shut down after two hours, even though thiamine was still present in the external environment (Praekelt et al., 1994). Thus the proteins of the transport pathway must be degraded very quickly if this control is due solely to repression.

Transcription of the thiamine transporter gene, THI10, was found to require the positive activation of Thi3p and was negatively controlled by TPP, but its expression was not affected by a thi2 mutation. This evidence supports the proposed model for thiamine regulation of (Nishimura et al., 1992b), Figure 1.9, which suggested that Thi3p but not Thi2p activates thiamine transport.

Positive regulation of the PH03 gene was found to be at the transcriptional level by PH06 (77/72) and PH07. The region responsible for thiamine sensitivity is located in the 5'- flanking region of the PH 0 3 gene at position -234 to -215 with respect to the ATG translation initiation site. Gel-shift assays showed that proteins from nuclear extracts of cells grown in thiamine-free medium bind to this region (Nosaka et al., 1992). Other genes regulated by TPP have a similar motif including, 77/74, 777/5. TH16 and THI80.

1.7.4 Regulation in other organismsIn S. pombe three mutations defining thiamine regulatory genes were identified

following a screen for constitutivity of T-rAPase activity, rnrl, tnr2 and tnr3 (Schweingruber et al., 1992). The tnr3 gene was defined genetically as a negative regulator of thiamine metabolism and was subsequently cloned and characterised (Fankhauser et al., 1995). It was found to encode the TPK enzyme, analogous to the THI80 gene in S. cerevisae. Although the genes defined by tnrl and mr2 have not yet been characterised it is known that the mutant strains are not only derepressed for pho4 (which encodes the T-rAPAse) but also for the biosynthetic genes thi2, thi3 and thi4, as well as the thiamine transport system. Thus, these genes are probably involved in negative regulation of thiamine metabolism in S. pombe, along with tnr3. In addition a positive thiamine regulatory gene, thil or ntfl, was isolated

25

and characterised from fission yeast by two different groups (Fankhauser & Schweingruber, 1994; Tang et al., 1994). Strains carrying mutations in the thil gene displayed little or no pho4, thi3 or thi2 message. Thus the thil mutation may be epistatic over tnrl, tnr2 and tnr3, leading to the suggestion that Thil protein functions downstream and interacts with the general transcription machinery (Schweingruber et al., 1992). The protein was found to contain an NH2 terminal Cys6 zinc finger motif, analogous to that found in the Gal4p transcription factor, indicating a role for thil, as a DNA-binding transcription factor for thiamine regulatable genes.

The TATA box of the widely used thi3 promoter has been studied and is located -25 to -30 bp with respect to the transcription start site. Using thi3 promoter deletions fused to the reporter gene chloramphenicol acetyltransferase (CAT), a putative thiamine activation element was identified. This sequence, located at 54 to 62bp upstream from the TATA box, is homologous to the binding site of a mammalian transcription factor, c/EBP, and was shown to bind proteins. However it is not known whether this sequence is specific for thiamine-repressible genes (Zurlinden & Schweingruber, 1997).

Perfectly conserved 1 lbp elements have been identified in the promoters of thi2 and thi3, located downstream from the TATA boxes and spanning the transcriptional start site (Manetti et a l, 1994). This element is not present in the pho4 promoter, indicating that this cis-acting element is not conserved between all thiamine-regulated genes. In the pho4 promoter a UAS sequence has been found which lies very close to the TATA box and is responsible not only for activation of gene expression but also for regulation by thiamine. This close association with the TATA box is thought to be important for the negative regulation by thiamine which appears to act post-transcriptionally. Positioning of the element does not affect the activation however, which is likely to be subject to control by the Thil protein (Silvestre & Jacobs, 1997).

In E. coli no genes involved in thiamine regulation have yet been isolated, although it is known that the biosynthetic genes are repressed at intracellular TPP concentrations of 35|iM. Some of the biosynthetic enzymes, such as HMP kinase, HMP-P kinase. HET-

kinase, thiamine phosphate synthase (TPS) and TP kinase have been shown to have reduced activities in the presence of thiamine (reviewed by Begley, 1996). Also a mutant has been identified which displays elevated levels of TPS and HET kinase in the presence of thiamine, but this has not yet been fully characterised (Kawasaki and Nose, 1969).

1.8 Aims of the projectDespite the fact that thiamine was the first vitamin to be isolated and that a great deal

of work has been done on the biochemical and genetic aspects of its biosynthesis and metabolism, especially in E. coli, there still remain a lot of unanswered questions. In this study, the model eukaryote, S. cerevisiae, was chosen as the organism in which to carry out

26

a genetic analysis of this system because of the ease of manipulation and our advanced understanding of its genetics; also because so much was still yet to be discovered with regard to its thiamine biosynthetic pathway. At the beginning of this work the genes encoding five out of the six downstream enzymes, in S. cerevisiae, had not been characterised, only two genes acting in the biosynthesis of the precursors had been isolated and two regulatory mutants had been described but their wild-type genes had not been characterised. My initial aim was to isolate novel genes involved in thiamine metabolism with the expectation that by elucidating their functions, our understanding of how this essential vitamin is synthesised would be advanced.

The approach I decided to take would incorporate a number of stages:1) Isolation of a set of thiamine auxotrophic strains, using more than one method of mutagenesis in order that the bias of mutations created would be minimised. The resultant mutants would be subjected to complementation analysis in order to elucidate how many genes were represented by them and also to find out if they were allelic to any of the known thiamine mutations. In addition the first isolated thiamine mutation, th il, was still uncharacterised, therefore I would include this auxotroph in the collection produced from mutagenesis for subsequent analyses.2) Physiological growth tests would be carried out in order to pinpoint which branch of the pathway was affected by each of the mutations.3) A representative mutant from each complementation group defining new genes would then be used for functional complementation with a yeast genomic library, since this appeared to be the easiest and most effective way to identify new genes. A low copy plasmid library made from S. cerevisiae genomic DNA was available for this purpose.4) The DNA sequence from any gene thus identified would be obtained, either by carrying out sequence analysis of the whole length of the ORF or by comparing a small length of sequence with the Saccharomyces Genome Database. The nucleotide sequence of these genes would be conceptually translated in order to obtain the amino acid sequences. Homologous proteins and motifs could then be identified by comparison with protein sequence databases.5) Assignment of a function to each gene isolated would be attempted by consideration of the site of action of the gene product and the function of any homologous proteins or motifs. Further, if possible, enzyme analysis of either the purified protein or crude extracts from cultures of wild-type and mutant yeast strains, would be carried out to confirm the deduced functions.

From this information it should be possible to identify the number of genes involved in thiamine metabolism in S. cerevisiae, their sites of action and possibly their functions. It may then be possible to develop new hypotheses for how these mechanisms are controlled.

27

CHAPTER TWO

Materials and Methods

2.1 Strains and plasmids2.1.1. Yeast strains (Saccharomyces cerevisiae)

Strain Genotype Source or reference

W303a Mata, ade2-l, canl-100, leu2-3-112, trpl-1, ura3-J, h is3-ll-115, R. Rothstein, Columbia

University.

W303a Mato., ade2-l, canl-100, leu2-3-112, trpl-1, ura3-l, his3-l 1-115, R. Rothstein, Columbia

University.

PMY3 Mata, ura3-52 P. A. Meacock

KBY4 Mata, ade2-l, canl-100, leu2-3-112, trpl-1, ura3-l This study

DG622 Mata, his3A200, ura3-167, leu2A, trplA, GAL I pGTy 1H3HIS3 (Guthrie & Fink, 1991)

W303A ilv2::

URA3

ade2-l, canl-100, leu2-3-J12, trpl-1, ura3-l, his3-l 1-115,

ilv2::URA3

This study

Mating type tester strains

MCPl M ata, his4 M. Pocklington

MCP6 Mata, his4 M. Pocklington

Thiamine mutant strains

KBY5a/a Mata/a, ade2-l, canl-100, leu2-3-112, trpl-1, ura3-l, his3-l 1-

115, thi4::URA3

This study

Y02587 Mata, thil, ural, trpl YGSC, Berkeley

Y025S7.URA Mata, thil, URAl, trp l, derived from Y02587 x W303apUP35 This study

KBY6 Mata, thil, ura3-52, trp l. derived from Y02587.URA x PMY3 This study

058-M5 Mata, thi2, gal4 (Kawasaki et al., 1990)

KBY7 Mata, thi2, gal4, ura3. derived from 058-M5 This studv

T49-2D Mata. thi3, leu2-3-l 12, ura3-52, trpl-A63. I\s2-801, his3-A200 (Nishimura et al.. 1992b)

PMY3 Athi4 M ata. ura3-52. Athi4::URA3 (Praekelt and Meacock.

1992)

W303Ar/z/2.v

URA3

ade2-l, canl-100, leu2-3-112, trpl-1, ura3-I, his3-J 1-115,

thi2::URA3

This study

W303Af/z/3::

URA3

ade2-l, canl-100, leu2-3-112, trpl-1, ura3-l, his3-l 1-115.

thi3::URA3

This study

UV1—>4 UV induced Thi* mutants derived from W303a.pUP35 This studv

Tyl-1—>140,

Ty 1-2—>3,

Tv3-1—>10

Ty induced Thi' mutants derived from DG622 This study

Table 2.1 Yeast strains used in this study

28

2.1.2 Bacterial strains (Escherichia coli)Strain Genotype Source or reference

NM522 F' lacHA(lacZ)M15,proA +B+/A(lac-proAB)thi, supE,

A( hsdMS-mcrB)5

(Gough & Murray, 1983)

XL 1-Blue recAl, endAl, gyrA96, thil, hsdR17, supE44, relLl, lac [F*

proAB, lacBZAM15, TnlO (tetr)l

(Bullock et al., 1987). Stratagene

JM110 rpsh, thr, leu, thi, lacY, galK, galT, ara, tonA, tsx, dam,

dcm, supEAA, A(lac-proAB) [F'fraD36proAB/<2cl9ZAM151

(Yanisch-Perron etal., 1985)

MH1578 F~t recAl, endAl, gyrA96, thr-1, SupE44, relAl, hsdR17

(r/F, mk+ ) rpsL

(Sedgwick & Morgan, 1994)

MH1599 recAl, endAl, gyrA96, thr-1, SupE44, relAl, hsdR17,

R388::Tn URA3

(Sedgwick and Morgan, 1994)

Table 2.2 Bacterial strains used in this study

2.1.3 Plasmids and vectors

Plasmid Genotype Source or reference

pUC18 / 19 lacZa+, ampr (Yanisch-Perron et al.,

1985)

YCp50 am pr, tetr, CEN4, ARS1, URA3 (Ma et al., 1987)

pUP35 ampr, CEN4, ARS1, URA3, tet::THI4-lacZ (fusion) (Praekelt and Meacock,

1992)

pRS315 ori, ampr, lacZ, CEN6, ARS4, LEU2 (Sikorski & Hieter,

1989)

pKB13 THI5 gene carried on YCp50 This study

pKB15 THI6 gene carried on YCp50 This study

pKB19(a—»h) ILV2 gene carried on YCp50 (plus deletions, described in text) This study

pKB20 THI3 gene carried on YCp50 This study

pKB21 THI3 gene carried on YCp50 This study

pKB22a URA3 gene carried on pUC19. with Xba\ sites at the 5’ and 3’ ends This studv

pKB22 ilv2A::URA3 cassette carried on YCp50 This studv

pKB23 ilv2A::URA3 cassette carried on pUC19 This study

pKB24 THU gene carried on YCp50 This studv

pKB25—»28 Hybrid plasmids of ILV2 and THI1 genes, as described in text. This studv

pKB29 Site-directed mutagenesis Spe 1 fragment carried on pUC19 This study

pKB30 Site-directed mutagenesis Spe 1 fragment carried on YCp50 This studv

pKMl THI2 gene carried on YCp50 This study

pKM2 THI3 gene carried on YCp50 This study

pMDl TH12 gene carried on YCp50 This study

Table 2.3 Bacterial and yeast plasmids used in this study

29

2.2 Growth media and conditionsFor solid medium agar was added to a final concentration of 2% (W/V).

2.2.1. Bacterial mediaBacterial strains were grown in Luria broth or Luria agar (Maniatis et al., 1982) and

incubated at 37°C. Antibiotics were added as follows: ampicillin, to a final concentration of 50jj,g/ml; methicillin to a final concentration of 50 p,g/ml; streptomycin to a final concentration of 1 0 0 jug/ml.

2.2.2. Yeast media and growth conditionsYeast strains were incubated at 28°C - 30°C, unless otherwise stated.Glucose was added as a carbon source to yeast media to a final concentration of 2%

(w/v), unless otherwise stated. Yeast was grown in Yeast Peptone Dextrose (YPD) (Sherman & Hicks, 1991) when no selection was required. For selective growth yeast was grown in SD media supplemented with the required amino acids and bases. Sporulation was carried out by growth on acetate based sporulation media, following growth on rich pre- sporulation medium, as described by Guthrie and Fink (1991).

To test for thiamine auxotrophy, yeast strains were grown in Wickerham's minimal medium (Wickerham, 1951), supplemented with appropriate amino acids and bases. This consisted of a salts mixture [KH2 PO4 (lg/1), MgS0 4 .7 H 2 0 (0.5g/l), NaCl (0.5g/l), CaCl2 -6 H2 0 (0.5g/l)], 2% (w/v) glucose as a carbon source, (NH4 )2 SC>4 (2.5g/l) as a nitrogen source, trace elements [H3 BO3 (8 |iM), MnS0 4 .4 H2 0 (2p.M), ZnS0 4 .7 H2 0

(IjiM), FeCl3.6H20 (lpM ), Na2Mo04-2H20 (ljiM), KI (lpM ), CUSO4 .5 H2 O (O.lpM)], and vitamins [nicotinic acid (65p,M), pantothenic acid (25|iM), pyridoxine (9jxM), inositol (IIOjiM), biotin (IjllM), p-aminobenzoic acid (4p.M), riboflavin (2jiM)]. Thiamine was added to the medium at either non-repressing concentrations (0 or 0.02pM to allow growth of thiamine auxotrophs) or repressing concentrations (2jiM). Thiamine precursors were added to Wickerham's medium to a final concentration of IOjiM for either HMP or HET.

2.3 Manipulations in yeast2.3.1 Low efficiency transformation

Yeast was transformed using the "one step" lithium acetate method as described by Chen et al. (Chen et al., 1992).Solutions:One-step buffer: 40% (w/v) PEG 3350, 0.2M Li acetate (pH5.0), 0.1M DTT Procedure:Yeast cells were grown overnight in 10ml YEPD. 500jil of culture was pelleted in a microfuge (13,000 rpm, 10 sec), resuspended in lOOp.1 one-step buffer and l-2 |ig

30

transforming DNA added. Samples were mixed well, incubated at 45°C for 30 min, then plated directly onto selective medium and incubated at 30°C for 2-3 days.

2.3.2 High efficiency transformationHigh efficiency transformations of yeast were carried out using lithium acetate,

single-stranded DNA and PEG, as described by Geitz et al. (Geitz et al., 1995).Solutions:TE: lOmM Tris-HCl (pH7.6), ImM EDTA (pH8.0)TEL: lOOmM Li acetate in TE (pH7.6)PEG: 40% PEG 3350 in TEL.Procedure:Yeast cells were grown overnight in 10ml YPD, diluted to a concentration of 5x10^ cells/ml in 20ml fresh YPD and re-grown to 2x10^ cells/ml. Cells were harvested at 3,000 rpm for 5 min, washed twice in sterile water, once in TEL then resuspended gently in 200pl TEL and incubated at 30°C for 15 mins. To 50j l l 1 cell aliquots was added l|ng transforming DNA, 5pl lOmg/ml single stranded salmon sperm DNA and 300pl freshly made PEG. Samples were

vortexed, incubated at 30°C for 30 min, heat shocked at 42°C for 20 min then plated onto selective medium and incubated at 30°C for 3-5 days.

2.3.3 Small scale plasmid preparations from S. cerevisiae Solutions:BME Buffer 0.9M Sorbitol

0.05M Na2P04 (pH 7.5)0.1 % P-mercaptoethanol (added just before use).

Procedure:Yeast cultures were grown up overnight in 10 ml of the appropriate selective media from a single colony. 4.5 ml of this culture was pelleted at 5000rpm and resuspended in 800 pi of BME buffer. 25 pi of 10 mg/ml yeast lytic enzyme was added, mixed by gentle inversion

and incubated at 37°C for 30-45 minutes until spheroplasts formed. Samples were then switched to 70°C for 20 minutes, followed by the addition of 200pl 5M potassium acetate

and incubation on ice for 45 minutes. The resulting precipitate was pelleted at 13000rpm for 10 seconds and the supernatant transferred to a fresh tube. 0.55 ml of isopropanol was added, the tube mixed and left at room temperature for 5 minutes. Plasmid DNA was then pelleted at 13000rpm for 10 minutes, washed in 70% ethanol and resuspended in 20 pi TE

buffer.

31

2.3.4 Plasmid curingFor the recovery of ura3 auxotrophs (e.g. from loss of a plasmid carrying the URA3

gene) yeast strains were grown on medium supplemented with lmg/ml 5-FOA and O.Olmg/ml of uracil to allow growth of ura3 strains (Guthrie and Fink, 1991). Segregation of plasmids carrying other nutritional markers was achieved by growing on non-selective medium (YPD) and testing for plasmid loss by replica-plating onto selective medium.

2.3.5 Preparation of highly purified genomic DNATotal chromosomal DNA of high quality, for use in Southern blot analysis and as a

template for Polymerase Chain Reactions (PCR), was prepared from spheroplasts using a method based on Cryer et al. (Cryer et a l, 1975).

Solutions:Solution A: 1.2M sorbitol, 25mM EDTA, pH8.0Solution B: 1.2M sorbitol, 0.1M Na citrate, lOmM EDTA, pH8.0Solution C: 3% sarkosyl, 0.5M Tris-HCl, 0.2M EDTA, pH7.6Procedure:Yeast cells grown overnight in 10ml YPD were diluted to 2x10^ cells/ml in 50ml YPD, re­grown to 2x10^ cells/ml and pelleted at 6000 rpm for 5 minutes. Cells were resuspended in 5ml of solution A, 175|il 1M DTT added, incubated at 30°C for 30 minutes with constant

agitation. The cells were repelleted (6000 rpm, 5 min) and resuspended in 5ml of solution B. 100|il lOmg/ml yeast lytic enzyme was added, samples were mixed and incubated at 30°C

for 30-45 min with constant agitation until cells were spheroplasting. After spheroplasting, cells were washed three times in 5ml of 1.2M sorbitol (pelleting each time at 5000 rpm, 5 min) and resuspended in 2ml of solution C. lOOjil 2mg/ml proteinase K (made up in solution

C) was added and cells incubated at 55°C for 1 hour. The volume was made up to 5ml with TE buffer and the lysate extracted three times with phenol/chloroform and twice with chloroform/iso-amyl alcohol until the interface was clear. Nucleic acids were precipitated in 2 volumes of cold ethanol for 15 min on ice and pelleted (5000 rpm, 5 min at 4°C). DNA/RNA was resuspended in 500pl TE buffer plus 20|il lOmg/ml RNase and incubation at 37°C for

1-2 hours. The DNA was reprecipitated in cold ethanol, washed twice in 70% cold ethanol, air dried and resuspended in 250-500|il TE buffer.

2.3.6 Gene disruptionFor disruption of wild-type loci the Rothstein "one-step disruption" method was used

(Rothstein, 1983). The cloned fragment containing the gene of interest was digested with a restriction endonuclease that cleaves within or just outside the gene and a fragment carrying a selectable yeast marker was ligated into the cut site. The disruption cassette was then liberated by digesting with a restriction endonuclease which cleaves within the flanking

32

DNA. Homology to the gene must be retained on both sides of the insert. The linear fragment was then transformed into a "wild-type" yeast strain and the cells were then plated onto selective medium. Substitution of the linear disrupted sequence for the resident chromosomal sequence should occur.

2.3.7 Gap repairGap repair was carried out as described by Orr-Weaver and Szostak. (Orr-Weaver &

Szostak, 1983). A replicating plasmid carrying a chromosomal ARS sequence and flanking DNA of the gene of interest, was digested with (a) restriction endonuclease(s) which cleave(s) just inside the gene or within the flanking DNA. After transformation into yeast, the double-strand gap of the plasmid was repaired by recombination with the homologous chromosomal sequence.

2.3.8 Making Y02587 ura3The THI1 wild-type gene was to be isolated using a low copy plasmid library,

constructed in the yeast vector YCp50. The thil mutant strain Y02587 therefore had to be made ura3. Since the strain was already uracil requiring {ural) it was first converted to uracil prototrophy by crossing with the Ura+ strain W303a.pUP35 and a th ill URA1 derivative

was isolated from the meiotic progeny. This strain Y02587 Ura+ {Mata, thil, trpl, URA1) was then crossed against a ura3 strain, PMY3 {Mata, ura3). The strain KBY6 {Mata, thil, trpl, ura3) was isolated from the meiotic products of this cross.

2.3.9 Making 058-M5 ura3The strain had to be ura3 in order to select for the transforming library clones. Since

this strain had no other nutritional markers it was not possible to cross it with a ura3 strain and select a th il ura3 derivative from the meiotic progeny. Instead the ura3 disruption construct AMO (obtained from M. Pocklington, Leicester University) was used. This

plasmid carries the URA3 gene with a 248bp fragment (189 - 437) deleted. The disruption cassette was released on a 925bp Sail fragment, isolated from an agarose gel following electrophoresis and transformed into the yeast strain 058-M5. Transformants were selected on SD medium containing 5-FOA and then purified. Gene replacement was confirmed by checking for uracil auxotrophy. A thil strain carrying the ura3A140 mutation was thus

created and was designated KBY7.

33

2.4 Manipulations in E. coli2.4.1 Transformation

Competent E. coli cells were prepared and transformation carried out using a method based on the original calcium chloride method of Mandel and Higa (Mandel & Higa, 1970). Plasmid DNA or DNA ligation mixes were added to IOOjllI competent cells and incubated on

ice for 30 min. Cells were then heat shocked at 42°C for 2 min, the volume made up to 1ml with LB and re-incubated at 37°C for 1 hour. Cells were pelleted in a microfiige (13000 rpm, 1 min) resuspended in lOOjil LB, plated onto selective medium and incubated at 37°C

overnight.

2.4.2 Small scale plasmid preparationsPlasmid DNA was isolated from 2ml culture of E. coli according to the protocol

described by Serghini et al (Serghini et al., 1989). Cells were disrupted by vortexing in a phenol solution. This was followed by spinning to get rid of cell debris, precipitating the nucleic acids and then treating with RNase enzyme.

2.4.3 Large scale plasmid preparationsSupercoiled plasmid DNA was isolated using QIAGEN-tip 100 columns (supplied

by Qiagen). The method, which combines the alkaline lysis method with ion exchange chromatography, was carried out according to the manufacturer's instructions.

2.4.4 Bacterial transposon mutagenesisIn order to identify the gene of interest within a genomic library clone, the bacterial

mutagenesis method of Sedgwick and Morgan (Sedgwick and Morgan, 1994) was employed. The principle behind this procedure is shown in Figure 2.1.

The ampicillin resistant target plasmid was transformed into into the streptomycin sensitive (Sms) strain, MH1599, carrying Tn1000 with the S. cerevisiae URA3 gene inserted into it, to produce a Ampr, Sms donor strain. The recipient strain was MH1578 (Amps, Smr). 10ml LB cultues of the donor and recipient strains were grown overnight from single colonies in the presence of the appropriate antibiotic. 0.1ml of each culture was used to inoculate separately 10ml LB medium and the cultures shaken at 37°C until an OD600 of 0.2 was reached. 1ml of donor bacteria was transferred to a sterile filter disc by

vacuum filtration and lml of recipient culture was transferred to the same disc. The disc was placed on a pre-warmed L agar plate and incubated at 37°C for lhour. The disc was then placed into a plastic sterilin tube containing lOmls of MgSC>4 and the cells dislodged by

vortexing vigorously for ~30secs. The disc was removed from the tube and the resulting cell suspension centrifuged (5000 rpm, for 5 min). The pellet was resuspended in lml lOmM M gS0 4 and 10|il and 100p.l aliquots were plated onto L agar containing methicillin (50p.g/ml), ampicillin (50pg/ml) and streptomycin (lOOjig/ml). Colonies growing on this

34

SmS Smr

Conjugativeplasmid

Tn TnTn

Targetplasmid

Am p

Amp

Conjugativeplasmid

TnTn Tn

Am p

Transposedplasmid

Tn

A B C > > - >■Donor Cell Mating Recipient Cell

Figure 2.1

Transposon mutagenesis using Tn1000.

A) Duplicative insertion links a conjugative plasmid carrying TnlOOO in a cointegrate with the target plasmid.

B) Conjugation transfers the cointegrate from the donor to the recipient cell.

C) Site-specific recombination between the two copies of the transposon in the cointegrate releases a transposed target plasmid and the original conjugative donor plasmid. After mating, selection with streptomycin eliminates the donor cells and with additional ampicillin selection (along with methicillin) the recipient cells are also killed, except for those which have received a transposed target plasmid through conjugation.Taken from Sedgwick and Morgan (1994).

medium would contain the target plasmid with the Tn1000 transposon randomly inserted into it.

2.5 DNA manipulations2.5.1 Restriction enzyme digests

Restriction enzymes were obtained from BRL Ltd or New England Biolabs (NEB) and digests were carried out in the reaction buffers provided, according to the manufacturers' instructions.

2.5.2 DNA agarose gel electrophoresisDNA fragments were separated by agarose gel electrophoresis, typically in 0.8%

(w/v) agarose in 1 x TAE buffer, containing 0.5|ig/ml ethidium bromide as described by

Maniatis et al. (Maniatis et a l, 1982). The separated DNA products were visualised under UV light.

2.5.3 Isolation of DNA fragmentsDNA fragments were recovered from agarose gels using the GENECLEAN II kit

(available from BIO 101 Inc) or the Wizard DNA Cleanup System (available from Promega), according to the instructions given by the supplier.

2.5.4 DNA dephosphorylationDephosphorylation of DNA was carried out using Calf Intestinal Alkaline

Phosphatase (supplied by Boehringer-Mannheim) or Shrimp Alkaline Phosphatase (supplied by Amersham), according to the manufacturer's instructions. Reactions were carried out in a total volume of 50fil, containing 1 unit phosphatase and were incubated at 37°C for 30-45

min. Dephosphorylated DNA was recovered by phenol extraction and ethanol precipitation.

2.5.5 DNA ligationT4 DNA ligase (Gibco-BRL, lU/jil) was used. Generally reactions were carried out

in a total volume of lOjxl, containing ljil ligase, 2|il T4 ligation buffer, plus DNA samples

being ligated. These were incubated overnight at 15°C. The amount of DNA used to obtain the optimal vectorrinsert ratio, according to Dugaiczyk et al (Dugaiczyk et a l, 1975), was calculated based on the relative lengths of the two DNA fragments.

2.5.6 Southern blottingFollowing electrophoresis genomic DNA was depurinated, denatured, neutralised

and then transferred from the agarose gel to a Hybond-N membrane, by capillary action

35

(Maniatis et al., 1982). The DNA was cross-linked to the filter membrane by UV illumination.

2.5.7 Hybridisation with a 32p radiolabelled probeRadiolabelled DNA probes were prepared by the random hexamer priming method

(Feinberg & Vogelstein, 1983) using a 32PdCTP. Filters were prehybridised and

hybridised in Church-Gilbert buffer (Church & Gilbert, 1984) at 65°C and then washed three times in 3 x SSC, 0.1% SDS solution at 65°C. The filters were wrapped in clingfilm and X- ray film was exposed to the radioactivity at -70°C, for the required amount of time to produce a signal.

2.5.8 Hybridisation with a non-radioactive probeA non-radioactive method for labelling DNA hybridisation probes was also utilised

(Gene Images, Amersham Life Sciences). The hybridisating fragment was denatured and single-stranded DNA was labelled with nucleotides including fluorescein-11-dUTP. The fluorescein groups could then be detected indirectly by binding an anti-fluorescein antibody conjugated to an alkaline phosphatase used to catalyse light production by enzymic decomposition of a stabilised dioxetane substrate. However more recently a method where DNA was directly labelled with a thermostable alkaline phosphatase enzyme was developed, negating the need to use an antibody and subsequent washes. All procedures were carried out according to the manufacture's instructions.

2.5.9 DNA sequencingPrimers for sequencingDNA cloned into the BamHl site in pBR322 DNA were

supplied by New England Biolabs - 1223 (clockwise) and 1219 (counter-clockwise). Custom-made primers were supplied by CMHT laboratory, Leicester University, as described in Table 2.4.

DNA was sequenced on an ABI model 373A DNA Sequencer, following sample preparation using the PRISM ™ Ready Reaction DyeDeoxy™ Terminator Cycle Sequencing Kit.Terminator premix: 1.58|iM A-DyeDeoxy, 94.74JJ.M T-DyeDeoxy, 0.42jiM G-DyeDeoxy, 47.37pM C-DyeDeoxy, 78.95^iM dITP, 15.79pM dATP, dCTP, dTTP, 168.42mM Tris- HC1 (pH9.0), 4 .2 ImM (NH4 )2 S0 4 , 42.1mM M gCb, 0.42 units/jxl AmpliTaq DNA polymerase.Procedure:Sequencing reactions were carried out in a total volume of 20jil (overlaid wih liquid paraffin) containing 8.0|il teminator premix, 30-500ng template DNA and 3.2pmol primer DNA. The

cycling reaction was carried out in a Hybaid Omni-E thermal cycler, and consisted of 25 cycles of a denaturation step (96°C, 30 sec), an annealing incubation (50°C, 15 sec) and an

36

extension incubation (60°C, 4 min). After PCR the reaction mix was isolated from under the paraffin layer by pipetting, made up to lOOjxl with water and extracted twice in phenol:H20:Chloroform (68:18:14). Extension products were precipitated with 15pl 2M Na acetate (pH4.5), 300j l l 1 100% ethanol, washed in 70% ethanol, then analysed on the DNA

Sequencer.

Name Sequence (5'—>3') Target site

5 AGGGGAACTGAGAGCTCTA Tn1000 LTR (86 to 68)

KB6 TTTACTTGTGGAAGCGCC THI6 ORF (-91 to -74)KB7 GCCTTGAGGACCCATGGG THI6 ORF (+474 to +457)KB9 CCCGGATCCCCTTTGAGCTAAGAGGAG ILV2 ORF * (-43 to -27)KB10 CCCGGATCCGAGAAAGAAGTCGTGTCC ILV2 ORF * (2356 to 2338)

KB11 GCCTGTACGCTTATGACG ILV2 ORF (2051 to 2033KB12 GCTTGAACGGCAGAACTC ILV2 ORF (1697 to 1679)KB13 CAGACCTCTCCTTAACTGG ILV2 ORF (1336 to 1318)KB 14 CTTGGACCATCTGCATGG ILV2 ORF (953 to 935)KB15 GGTTGCCAACGACACAGG ILV2 ORF (1446 to 1463)KB 16 CAGAAGGCTACGC CAGAG ILV2 ORF (437 to 454)KB17 CCAGTCGCACACAAGATG ILV2 ORF (830 to 847)KB18 GCCAATGGTGGCAGGTGG ILV2 ORF (1956 to 1973)KB19 TCCAATGGCAGAGGCCTTTGCAGAC ILV2 ORF t (516 to 540)KB20 GTCTGCAAAGGCCTCTGCCATTGGA ILV2 ORF t (540 to 516)KB21 GTATTTCTCTTATCTGGTTG ILV2 ORF (-289 to -269)KB 22 GTCAGCCTCTTGGAAAGC ILV2 ORF (615 to 599)

Table 2.4 List of synthetic oligonucleotides used throughout this study* BamHl restriction enzyme site incorporated at the 5' end t Mismatch incorporated in the middle of the sequence

2.5.10 Polymerase chain reactionPCR was used for the amplification of DNA fragments and was performed using

either a Techne PHC-3 or a Hybaid Omn-E thermal cycler on 20j l l 1 reactions (overlaid with

liquid paraffin) containing:

37

dNTPs (250pM concentration of each dNTP)

primer DNA (500ng each primer)Template DNA (50-100ng for plasmid, 400-600ng genomic template)Deep vent polymerase (1 unit, NEB)Deep vent reaction buffer (1 x NEB)A typical reaction profile consisted of a single 95°C 5 min step, followed by 25

cycles of a denaturation step (95°C, 30 sec), an annealing incubation (50-60°C, 30 sec) and an extension incubation (72°C). The length of the extension incubation time was calculated using a rule of 1 minute per kb DNA being amplified. The products of PCR reactions were visualised following agarose gel electrophoresis.

2.6 Mutagenesis2.6.1 UV mutagenesis

The protocol followed was based on that described by Lawrence (Lawrence, 1991). Yeast cells (strain W303a.pUP35) were grown overnight in lOmls of Wickerham's medium containing ljiM thiamine and supplemented with tryptophan, histidine, leucine and adenine.

The cell density was calculated using a haemocytometer. The cells were washed twice in sterile water and resuspended in 20mls 0.9% KC1. Mutagenesis was carried out using a dose of 0.5 J/m^/sec whilst the cells were continuously stirred with a magnetic stirrer in a petri dish. In order to achieve a survival rate of approximately 50%, the cells were mutagenised for 2.5 minutes.

Following mutagenesis, the culture was split into 20 tubes (in order to avoid isolating many duplicates) and grown overnight in Wickerham's medium containing 0.1 pM thiamine

and supplemented with tryptophan, histidine, leucine and adenine.

2.6.2 Ty insertion mutagenesisThe protocol for Ty insertion mutagenesis is described in A Guide to Yeast Genetics

and Molecular Biology [Chapter 23] (Guthrie and Fink. 1991). Ty insertion mutagenesis was carried out using the plasmid pGTylH3///S3. This plasmid contains the Tyl element Ty 1H3 under the control of the GAL1 promoter, with the HIS 3 gene inserted into the BglU site. It also contains the URA3 gene for selection and a 2p ORI sequence ensuring a high

copy number of the plasmid. This plasmid was transformed into the yeast strain DG622. Transposition was induced by growth on SD plates containing galactose as the carbon source, at 15°C for 5 days. The medium also contained tryptophan, leucine and uracil but lacked histidine so that selection for a His+ phenotype was maintained during transposition (i.e. selection for the marked Ty element); uracil was included to allow for segregation of the plasmid.

38

After mutagenesis the cells were grown overnight in YPD broth in 10 separate tubes, to allow for segregation of the plasmid.

2.6.3 Site-directed mutagenesisSite-directed mutagenesis involved a series of a PCR reactions, described in detail in

Chapter 5.

2.6.4 Nystatin enrichmentNystatin enrichment was carried out as described by Snow (Snow, 1966). In order

for enrichment to occur the thiamine auxotrophs needed to be growth arrested. Therefore the enrichment process was carried out in thiamine-free medium. Firstly, the cells were starved for nitrogen for 4 hours, by growing in Wickerham’s minimal medium lacking ammonium sulphate supplemented with appropriate amino acids and bases. The cells were then grown in medium containing nitrogen for 4 hours to allow recovery, before nystatin was added to the medium, to a final concentration of 10pg/ml. The cells were grown in the presence of

nystatin for one hour, during which time the actively dividing cells (in this case the thiamine prototrophs) took up the antibiotic, where it disrupted the cell walls and killed the growing yeast. The arrested cells however did not readily take up the drug and these survived at a much higher rate. Thus enrichment for the arrested thiamine auxotrophs was achieved.

2.7 Analysis of mutants2.7.1 Dominance / recessiveness test against wild-type

Each mutant strain was crossed with a 'wild-type' strain (i.e. a thiamine prototroph), of the opposite mating type carrying appropriate genetic markers so that diploids could be selected. Mutants UV1-4 of strain W303.pUP35 were crossed with KBY4 (W303oc HIS3). Heterozygous diploids (His+, Ura+) formed were selected on SD plates containing trytophan. leucine and adenine and lacking histidine and uracil. The Ty insertion mutants of strain DG622 were crossed with W303a.pUP35. Heterozygous diploids formed were selected on SD plates containing trytophan and leucine and lacking adenine, histidine and uracil.

The heterozygous diploids were tested for thiamine prototrophy by replica-plating onto thiamine - free Wickerham’s media two to three times, in order to deplete the internal pool of thiamine, so that the mutant phenotype could be observed.

2.7.2 Sporulation of diploids and dissection of tetrads.After growth on sporulation medium for ~5 days the diploids were examined under

the microscope to see if tetrads had formed. If so the cells were resuspended in 100 pi of water to which 3pl of p-glucuronidase was added. The mixture was incubated at room

39

temperature for 5 minutes to digest the asci walls without dissociating the four spores from their ascus. Meiotic tetrads were then dissected using a Singer MSM Micromanipulator and the spores deposited onto YPD plates which had been poured on a levelling platform. The spores were incubated at 30°C for three days to allow germination and growth before the resultant colonies were replica-plated onto selective media.

2.8 Enzyme assays2.8.1 Preparation of permeabilised cells

Yeast strains were grown overnight to a concentration of 1 - 3 x 107 cells ml-1, in Wickerham's minimal medium supplemented with tryptophan, histidine, adenine, isoleucine and valine. Keeping everything at 4°C, cells were harvested at 5000 rpm for 5 minutes and washed twice in sterile distilled H2O. The cell were resuspended in 0.1 mM TRIS buffer (pH7.5), 0.1M NaCl, O.lmM EDTA plus protease inhibitors chymotrypsin (lmg/ml), PMSF (ImM) and pepstatin (lpM). They were permeabilised by adding lOOpl chloroform,

vortexing for 30 secs, collecting by centrifugation and the supernatant decanted off. Crude protein concentrations were measured using the Biorad "Bradford Reagent" method, according to the manufacturers' instructions.

2.8.2 Acetohydroxyacid synthetase (AHAS) assayAHAS was assayed via its ALS activity. Acetolactate production was assayed at 30°C

for 30mins, using a method based that of Ryan and Kohlhaw (Ryan & Kohlhaw, 1974). For each assay two tubes were set up, A and B, each containing a mixture of 40mM sodium pyruvate, lOmM MgCh, lOOmM TRIS-HC1 (pH7.5) and varying concentrations of TPP (0 - 200pM), to a total volume of lOOpl. The reaction was started by the addition of ~0.5mg of protein extract to each tube and was terminated by the addition of 50pl of 6N sulphuric acid to tube A and 150|il of 6N sodium hydroxide to tube B. The amount of acetolactate was

determined by converting AL to acetoin, measuring the total amount of acetoin produced (tube A) and subtracting the amount of background acetoin produced by the cells (tube B). Acetolactate is converted into acetoin by a decarboxylation step which involves heating the sample in an acid environment. Therefore tube A was incubated at 60°C for 30 minutes after which the sample was made basic by the addition of lOOjil of 12N sodium hydroxide.

Acetoin was measured according to the method of Westerfeld (Westerfeld, 1945). Briefly, each sample was made up to a total volume of lml with sterile distilled water, then 200JJ.1 of 0.5% creatine was added followed by 200jll1 of 5% a-naphthol (freshly made up in 2.5 N

sodium hydroxide) and the solutions mixed by inverting the tubes several times. The colour was allowed to develop for one hour at room temperature, the samples were centrifuged for 2 mins and then read on a spectrophotometre at 530nm against a blank made up of lml of water, 200|il of creatine and 200pl of a-naphthol.

40

2.8.3 Assay for P-galactosidase activity in liquid culturep-galactosidase assays were carried out using the method of Reynolds (Reynolds,

1989). The p-galactosidase activity of the wild-type strain W303a.pUP35 grown in the presence (2 |lIM ) and absence of thiamine was always measured as a control.

Solutions:Z Buffer: 60mM Na2 HPC>4 , 40mM NaH2 PC>4 , 10mM KC1, ImM MgS0 4 , 50mM p-mercaptoethanol (added just before use), pH7.0ONPG: 4mg/ml o-nitrophenol-P-D-galactoside in lOOmM KPO4 , pH7.0Procedure:Yeast strains containing promoter-/acZ reporter gene constructs were grown overnight in 10ml Wickerhams minimal medium with or without thiamine, until a cell density of 5x10^ - 107 cells/ml was reached. At this point OD600 values of the cultures were recorded, lml

aliquots pelleted in a microfuge (6000 rpm for 5 minutes), the cells washed in water and resuspended in lml Z Buffer. 50-IOOjj.I of this cell suspension was transferred to a fresh tube, the volume made up to 0.9ml with Z buffer, followed by addition of 45p.l chloroform and 5pl 0.1% SDS. Samples were vortexed for 15 sec using a multi-tube vortex adapter and incubated at 30°C for 15 min to equilibrate. 180jil ONPG was added to each tube, mixed by

inversion, re-incubated at 30°C and timing begun. Reactions were stopped by the addition of 450|il 1M Na2C0 3 when the solutions had turned yellow and the time noted. Samples were centrifuged (13000 rpm for 2 minutes) and the OD4 2 0 of the supernatant measured compared to a Z Buffer/ONPG blank, p-galactosidase activity values were calculated using

the following equation:UNITS = 1000 x OD4 2 0 / T x V x OD600

where T = reaction time at 30°C (min)V = volume of cells used (ml)

41

CHAPTER THREE

Isolation and characterisation of thiamine auxotrophic mutants

3.1 IntroductionA primary objective of this work was to create a collection of thiamine auxotrophic

mutants in the yeast S. cerevisiae, so that a comprehensive genetic analysis of its thiamine biosynthesis and metabolism could be carried out. To date no such systematic analysis has been conducted and the number of genes involved in the pathway remains unresolved. By determining the number of complementation groups represented amongst the mutant strains the number of genes involved in thiamine metabolism could be elucidated.

Two methods of mutagenesis were chosen: UV and transposon (Ty) insertion mutagenesis. Non-ionising radiation produced by ultra-violet light induces pyrimidine dimers in DNA which often results in transitional mutations (Suzuki etal., 1989). Therefore UV mutagenesis would be useful in creating point mutations which may lead to an altered or truncated gene product. Alternatively, promoter mutations may arise leading to up- or down- regulation of relevant genes. Mutations caused by insertion of Ty elements, on the other hand, produce disruptions leading mostly to null alleles and possibly cleaner auxotrophic phenotypes.

Ty elements are a family of transposons found in the S. cerevisiae genome in approximately 30-40 copies which constitute 5-10% of the total mRNA in a haploid yeast cell. The elements are ~6kb in length and comprise a large central region epsilon (e), flanked by direct repeats called delta (8) elements, where transcriptional initiation and termination

occur (Boeke et al., 1985). Reciprocal recombination and gene conversion between two Ty elements at different loci within the genome can lead to chromosomal aberrations such as deletions, inversions, duplications and translocations. Ty elements can also insert themselves into non-homologous target sequences by transposition, although it has been shown that transposition of Tyl elements is non-random and that there may indeed be some target site specificity (Natsoulis et al., 1989). It was also found that selectable genes could be inserted into the non-essential part of the coding sequence of Tyl elements and transposition would still occur. For example HIS3, expressed from within a Ty element, could complement the corresponding auxotrophic yeast mutant, such that a gene disrupted by insertion of this marked element, could then be followed genetically. A method for tagged Ty insertion mutagenesis was developed by using marked Ty elements fused to the controllable yeast GAL1 promoter, on a high copy plasmid eg.pGTyl-H3///53 (Garfinkel et al., 1988). Growth of yeast strains carrying a pGTy plasmid on galactose would induce high levels of transposition.

42

3.2 M utagenesis3.2.1 UV mutagenesis

Three UV mutagenesis procedures were carried out on the parent strain W303a.pUP35. Exponentially growing cultures of cells which had grown overnight in minimal medium supplemented with ljiM thiamine were taken and exposed to UV

irradiation. Mutagenesis was carried out using a dose rate of 0.5 J/m2/sec whilst the cells were continuously agitated with a magnetic stirrer in a petri dish. In order to achieve a survival rate of approximately 50%, the cells were exposed for 2.5 minutes. The survival rate for the cells following each screen was calculated. These were:

Screen I 55% survivalScreen II 35% survivalScreen III 50% survivalAfter mutagenesis the cells were distributed between twenty tubes so that by

choosing only one mutant from each tube, the chances of recovering mutants arising from independent events was maximised. After overnight growth in rich medium cultures were subjected to nystatin treatment in the absence of thiamine in order to enrich for mutants auxotrophic for that vitamin. Nystatin enrichment was carried out as described in Chapter 2. Treated cultures were plated onto minimal medium containing O.IjllM thiamine and

auxotrophs identified by replica-plating onto thiamine-free medium. Because of the residual amount of thiamine in the cells, it was necessary to carry out the replica-plating onto medium lacking thiamine two or three times before the auxotrophic phenotype was clearly detectable. Thiamine auxotrophs were thus identified and isolated.

From a total of approximately lxlO10 cells mutagenised 1.2 x 103 colonies were recovered after nystatin enrichment and four thiamine auxotrophs were isolated. These were designated UV1, UV2, UV3 and UV4.

3.2.2 Ty mutagenesisThree Ty insertion mutagenesis procedures were carried out on the parent strain

DG622 which comprised a functional GAL system and had been transformed with the plasmid bearing the transposable element pGTylH3///S3 . This plasmid contains the Tyl element Ty 1H3 under the control of the GAL1 promoter, with the HIS3 gene inserted into a unique Bglll site. The plasmid also carries the URA3 gene for selection and a 2p ORI

sequence to ensure a high copy number of the plasmid. Transposition was induced by growth on SD plates containing galactose as the carbon source at 15°C for 5 days. The medium also contained tryptophan, leucine and uracil, so that selection for a His+ phenotype was maintained during transposition (i.e. selection for the marked Ty element) and uracil was available to allow for segregation of the plasmid.

After mutagenesis the cells were divided among ten tubes and grown overnight in YPD broth. Nystatin enrichment was carried out the next day. The cells were then plated

43

onto Wickerham's minimal medium containing tryptophan, leucine, uracil and O.ljiM

thiamine. After 3-5 days' growth at 30°C colonies were picked onto the same medium and replica-plated onto medium lacking thiamine in order to identify any thiamine auxotrophs.

If a marked Ty insertion had occurred then the strain would retain a His+ phenotype after loss of the plasmid bearing the Ty element. Therefore in order to find out if the thiamine auxotrophs arose due to insertions of marked elements, the pGTyl-H7S3 plasmid was allowed to segregate out by growth in non-selective medium. Plasmid-free segregants were identified by their Ura- phenotype. It was not necessary to use 5-FOA to select for the segregants since the plasmid was found to be quite unstable, particularly after transposition had been induced. All the ura- segregants were also His+, indicating that a marked Ty element had inserted into the genome in each case.

Transposition efficiency of the population was measured after loss of the plasmid, as the fraction of Ura- colonies that were His+ i.e. the fraction of colonies in which the marked Ty element had integrated into the genome; this was found to be 32 / 72 = 44%. The reversion rate of the A his locus is expected to be zero, since the mutation was created

through a deletion.

Screen I Approximately 3xl07 cells were mutated and from 7 x 102 colonies recovered after nystatin enrichment 160 auxotrophs were isolated; these were designated Tyl-1 to Tyl- 160. Nystatin treatment enriched the percentage of thiamine auxotrophs in the population to 23% of total Ty insertion derivatives. In this screen, following mutagenesis the cell culture was not split into separate tubes before overnight growth and nystatin enrichment. The cells went through approximately twenty generations during this time, therefore some of the auxotrophs isolated in this screen are likely to be duplicates of each other.

Screen II Approximately lxlO6 cells were mutated, 5 x 102 colonies were screened after nystatin enrichment and three auxotrophs were isolated. These were designated Ty2-1, Ty2-2 and Ty2-3 (all from different tubes). Treatment by nystatin enriched thiamine auxotrophs to 0.6% of total population.Screen III Approximately l .lx l0 6 cells were mutated, 9 x 102 colonies were screened after nystatin enrichment and eight thiamine auxotrophs were isolated. These were designated Ty3-1, 2, 3, 4, 5, 7, 8. 9. Treatment by nystatin enriched thiamine auxotrophs to 0.88% of total mutants in the population. However, four of these, Ty3-1 to Ty3-4, were from the same tube; also Ty3-7 and Ty3-8 were from the same tube. Therefore these eight mutants may define only four independent mutational events.

Thus a total of 171 Ty insertion mutants were isolated. From the frequency of occurrence it is possible to calculate that these must represent at least eight different mutational events, probably more. The number of complementation groups defined by these and the four UV induced mutations was investigated.

44

3.3 Genetic analysisIn order to be able to carry out a complementation analysis it was first necessary to

establish whether the mutations were recessive or dominant.

3.3.1 Dominance / recessiveness testThe four UV mutants were crossed against KBY4 and the 171 Ty mutants were

crossed against W303a.pUP35 to form heterozygous diploids. In every case each diploid formed was prototrophic for thiamine; therefore all the mutations were recessive suggesting that they caused loss of a particular function.

3.3.2 M eiotic segregation analysisHeterozygous diploids of the four UV mutants and 29 of the 171 Ty insertion

mutants were induced to undergo sporulation and the resulting 4 spore asci were dissected and analysed. Segregation of the Thi" :Thi+ alleles was monitored, as were pairs of alleles of other genes, such as MAT, ADE2 and URA3, which were expected to segregate 2:2.

In the cases of the mutants, UV1, UV2 and UV3, sporulation of the heterozygous diploids and germination of the haploid progeny was difficult. The UV 1 haploid strain grew poorly and in all of seventeen tetrads dissected from the heterozygous diploid only two of the spores were viable; all of these were prototrophic for thiamine, implying that the allele causing thiamine auxotrophy was also responsible for the inability of the other spores to germinate. Growth of the mutant UV1 was so poor that further analysis of its thiamine mutation was impossible.

The haploid strain carrying the uv2 mutation also grew poorly. Approximately forty asci were dissected following sporulation of the heterozygous diploid; only four tetrads gave four viable spores. However segregation of the thi~:thi+ alleles displayed 2:2 segregation in each case, confirming that uv2 is a single locus mutation. In every case where only two or three viable spores were recovered, two of the colonies were found to be thiamine prototrophic. Again this implies that the thiamine auxotrophy is linked to a difficulty in spore germination. The UV3 haploid strain did not have the same growth defects as UV 1 and UV2 but germination of spores recovered following dissection of asci from the heterozygous diploid was poor. From approximately thirty tetrads dissected only five gave four viable spores; all of these tetrads showed 2 2 segregation of Thi':Thi+ phenotypes. The uv3 mutation is therefore a single locus mutation. All tetrads analysed displayed 2:2 segregation of pairs of alleles for the ADE2 and MAT genes, indicative of normal meioses.

In the case of the heterozygous diploids of UV4 and the twenty nine Ty insertion mutants analysed, sporulation and spore germination was much easier, in each case about half of the tetrads dissected produced four viable spores. All sporulated diploids showed 2:2

45

segregation of uracil or adenine auxotrophy rprototrophy and mating-type a:a, indicating that

normal segregation of alleles at meiosis had taken place. The Thi':Thi+ alleles were also found to segregate 2:2 in every case.

Thus despite the difficulties outlined it was shown that, apart from UV1, all mutants tested carried a mutation in a single gene.

3.3.3 Cosegregation of His+ and Thi- alleles.If the Ty induced thi' mutations arose through insertion of the TyHIS3 element into a

THI gene, then the thi' and HIS3 phenotypes should be coinherited and show extremely tight linkage. However, an excess of Tetratypes and Non-Parental Ditypes (His+Thi+ / His' Thi') over Parental Ditypes (His+Thi“ / His'Thi"1") was found, in most cases. Parental Ditypes occur when there has been no recombination between the two loci under study, in this case the thi and HIS3 genes. Tetratypes occur when there has been one crossover, and Non-Parental Ditypes occur when there has been more than one cross-over event between the alleles. If the Tyl-H3 construct had inserted into the thiamine gene causing the mutation then it would be impossible to obtain Tetratypes and Non-Parental ditypes as the thi and HIS3 alleles would be tightly linked. Since the data indicated that they are not tightly linked then insertion of the T y l-HIS3 element did'not cause the mutation. The most likely explanation is that the marked Ty element had inserted somewhere else in the genome (thereby conferring the His+ phenotype on the strain) and a native Ty element has inserted into the thiamine gene causing its mutation. It has been reported that the frequency of transposition of native Ty elements is increased when transposition of a pGTy element has been induced, by growth on galactose containing medium, due to the overproduction of Ty mRNA. This may be caused by the elevated levels of one or more of the gene products of the marked Ty, TyA and TyB, catalysing the increased transposition of the chromosomal elements (Boeke, et al., 1985). It might have been possible to suppress this effect by using a spt3 strain, as SPT3 is required for transcription of the native Ty elements but not elements under the control of an inducible promoter, such as pGTy 1-H3HIS3 (Boeke et al., 1986). However the SPT3 gene also affects diploid formation and sporulation, making subsequent genetic analysis difficult in a spt3 strain (Winston et al.. 1984).

The thiamine auxotrophic phenotypes may alternatively have arisen due to fortuitous spontanteous mutations, or marked Ty elements integrating into the genes and then excising subsequently through recombinational events between the terminal 5 sequences. Such a

mechanism would remove the HIS3 gene but leave the THI gene disrupted by the single remaining 5 element. However gross chromosomal rearrangements such as inversions and

deletions may also arise through this type of recombinational event.Examination of the meiotic tetrads showed that the HIS3 and his3 alleles did not

segregate 2:2 in most cases; sometimes it was 3:1 and sometimes 4:0, suggesting that more than one marked Ty element had inserted into the genome. This may have been avoided by

46

reducing the induction time for transposition, and thereby reducing the number of insertions of both native and marked Ty elements. Nonetheless thiamine auxotrophs were isolated which was the main objective of this work.

3.4 Complementation analysisIn order to find out how many genes were represented by the thiamine mutations,

complementation analysis was carried out. Haploid segregants of both mating types carrying the thiamine mutation and appropriate nutritional markers were recovered from tetrad analyses. These mutants could then be crossed against each other, and the resulting diploids tested for thiamine auxotrophy; failure to complement would show that the mutations belonged to the same complementation group. The mutants were also crossed against other thiamine auxotrophic strains which were available in the laboratory, Y0587 {thil), 058-M5 (thi2) and KBY5 (thi4). Later the thi3 strain, T49-2D, was obtained; therefore this strain was used in some but not all of the complementation tests. The results of this analysis are summarised in Table 3.1 and Table 3.2.

During the course of this analysis it became apparent that most of the Ty insertion mutations, 141 out of 171, had occurred in THI4 since they did not complement KBY5 {thi4::URA3). These mutations, designated thi4-tyl, represent at least four different mutational events. The remaining thirty Ty insertion mutants and the four UV mutants were able to complement KBY5 and therefore carry mutations in thiamine genes other than THI4.

Of the twenty eight Ty insertion mutants from screen I which were not allelic with thi4 , twenty seven (Tyl-3, -15, -19, -24, -25, -26, -32, -36, -48, -53, -71, -87, -96, -97, -106, -116, -121, -122, -127, -129, -134, -135, -140, -141, -145 & -158) failed to complement each other. Therefore these auxotrophs must have mutations in the same gene. Although MATa strains were isolated from only eight of these and crossed against the others, it is reasonable to assume that the other nineteen would have given the same result. This complementation group will hence be referred to as Tyl-3.

Mutations in Tyl-3 did complement ty l-128, t \3 - l , t}'3-7, uv2, uv3, uv4, thil, thi2 and thi4\ these all represent separate complementation groups. The Ty insertion mutants from screen III which were not allelic with thi4, ty3-l or ty3-7, complemented each other, as well as ty l -3 , t y l -128, uv2, uv3, uv4, th il, thi2, thi3 and, of course, thi4-tyl. Thus the Ty insertion mutants appear to define five complementation groups, thi4-tyl, tyl-3, ty l -128, ty3-l & ty3-7. The UV mutants defined three distinct complementation groups, uv2, uv3 & uv4.

From the complementation analysis it can be observed that ten complementation groups (genes) are represented amongst the set of mutants (Table 3.2). Group 1 has 142 members and since this group also includes the original thi4::URA3 mutant the mutations must define the TH14 gene. Group 2, defined by tyl-3, has 27 members although it is not

47

Table 3.1

Complementation analysis of thiamine auxotrophs

Haploid strains carrying various thi mutations as indicated, were crossed and the resultant heterozygous diploids tested for growth on minimal medium lacking thiamine. If the diploid was found to be a thiamine auxotroph (denoted by -) the mutations were assigned to the same complementation group; if it was found to be a prototroph (denoted by +) they were assigned to different complementation groups.

* represents a set of 141 mutants all of which behave in the same way t represents a set of 27 mutants all of which behave in the same way

nd - not determined

1 2 3 4 5 6 7 8 9 10 11 12 Groupthi4- tyl-3] tyl-128 ty3-l ty3-7 uv2 uv3 uv4 thil thi2 thi3 thi4:: Mutationtyl* URA3

- + + + + + + + + + nd - thi4-tyl* 1- + + + + + + + + + + tyl-31 2

- + + + + + + + - + tyl-128 3- + + + + + + nd + ty3-l 4

- + + + + + + + ty3-7 5- - + + + + + uv2 6

- + + + + + uv3 7- + + + + uv4 8

- nd + nd thil 9- + nd thi2 10

- nd thi3 11- thi4::URA3 12

Table 3.2

Complementation groups of the thiamine auxotrophic mutants.

Complementation group Number of members Gene defined

1 142 T H U

2 27 TY1-3

3 2 THI3

4 1 TY3-1

5 1 TY3-7

6 1 UV2

7 1 UV3

8 1 UV4

9 1 THI1

10 1 THI2

The new mutants produced in this study were assigned to complementation groups, along with known thiamine auxotrophs, following complementation analysis. The number of mutants represented by each complementation group and the gene defined in each case is shown; known thiamine genes are shown in bold type.

known if these arose through one or more mutational event(s). Group 3 (tyl-128 ) contains two mutants one of which was T49-2D (thi3), therefore these must define the THI3 gene. Groups 4 to 8, defined by ty3-l, ty3-7, uv2, uv3 and uv4, respectively, contained only one member each; these were all in different complementation groups from those defined by the previously known thiamine mutations. Thus of the eight complementation groups represented by the set of UV and Ty induced mutants, two corresponded to the genes defined by the known thiamine mutations - thi3 and thi4; the other six appeared to define novel thiamine genes which were distinct from thil and thi2 (groups 9 and 10).

3.5 Growth phenotypesGrowth tests were carried out in order to find out which step in the pathway was

blocked, for each mutation. Thus a representative of each mutant type was tested for growth on minimal medium supplemented either with thiamine or with one of the precursors (HET and HMP) or the two together. Growth on HET would suggest that the mutation inactivates a gene that acts upstream of that precursor, whereas growth on HMP would suggest that the mutation blocks synthesis of the pyrimidine precursor. Growth on neither precursor would suggest that the gene which has been mutated acts downstream from the precursors, either in the condensation, phosphorylation or dephosphorylation steps. Alternatively the strain may carry a mutation in a positive regulatory function needed for expression of these genes. Rescue by addition of both precursors together would suggest that a regulatory gene affecting upstream functions, has been mutated. The results of this study are summarised in Table 3.3. As expected the thi4::URA3 mutant was able to grow in medium lacking thiamine but supplemented with HET, confirming that THI4 is indeed involved in the thiazole biosynthesis. All Ty mutations in the same complementation group, defined by thi4-tyl. showed the same growth phenotype.

The growth defects of strains Y02587 (th il), Tyl-128, Ty3-7 and UV3 were restored by the addition of HMP to the medium, suggesting that they are all blocked in pyrimidine precursor synthesis. The THI5 gene family (comprising 777/5, 777///, THI12 and THI13) is thought to function in this branch of the pathway but since this gene is present in four copies in the 5. cerevisiae genome it is unlikely that mutation of a single THI5 gene would display thiamine auxotrophy; infact it is known that gene knockouts at the 777/5 and THI12 loci do not create thiamine auxotrophic phenotypes (Hather, 1996). A strain disrupted in all four genes was not available, so complementation analysis was not possible and even if it were, the analysis would be very complicated. However the THIS gene was available on a low copy plasmid, pKB13, which was transformed into these four HMP rescued strains to see if it was able to rescue their growth defect. In all cases the THIS gene did not complement the mutation. From this result it can be concluded that there are potentially five

48

Table 3.3

Growth requirements of thiamine mutant strains.

Mutation Minimal medium

Noaddition

+HMP +HET +HET & HMP

+THI

thi4::URA3 - - + + +

thi4-tyl - - + + +

thil - + - + +

tyl-128 - + - + +

ty3-7 - + - + +

uv3+

- + +

thi2 - - +

uv4 - - +

tyl-3 -"

_ +

ty3-l * - +

uv2 - + +

thi3 - _ - + +

wild-type + + + + +

Cultures of thiamine mutant strains were streaked onto Wickerham's minimal medium supplemented with either thiamine, HMP, HET or HET & HMP (to a final concentration of 2pM). The strains were also tested for growth in the absence of thiamine or precursors.

Growth was assessed after incubation at 30°C for 3-4 days.+ indicates growth, - indicates no growth

different functions carried out by the genes acting in the biosynthesis of HMP: 77/77, Tyl- 128, Ty3-7, UV3 and the THIS gene family.

Four complementation groups defined by mutations thi2, uv4, ty l-3 and ty3-l appeared to act downstream from the precursors since the presence of the precursors in the medium did not correct the thiamine auxotrophies. These strains may carry defects in structural genes for the characterised downstream enzymes, the uncharacterised phosphatase or putative regulatory genes.

The uv2 mutant, along with the thi3 strain, T49-2D, was rescued by the addition of both precursors to the medium but not either separately. This indicated that the uv2 strain is defective in a positive regulatory function. Since this strain was found to complement mutations in both the thi2 and thi3 mutants strains, uv2 may define a novel thiamine regulatory gene.

3.6 Further analysis of uv2 and uv3.Because of the interesting phenotype of the strain UV2, the idea that it may encode a

regulatory function was investigated further. The transcriptional activities of the thiamine regulated genes THI4, THI5 and 77/7(5 (see -Chapter 6) within a uv2 background was studied. Promoter- LacZ constructs of these genes had already been made: THI4-LacZ = pUP35 (Praekelt et al., 1994); THI5-lacZ = pRB3 and THI12-LacZ = pRB4 (Burrows, 1997); THI6-LacZ = pLK2 (Kew, 1996). These plasmid constructs were transformed into the UV2 strain and the wild-type strain, W303. Assays measuring p-galactosidase activity

were carried out on the tranformants, following growth in minimal medium containing repressing (2.0}iM) or non-repressing concentrations (0.02pM) of thiamine. The assays

could not be carried out after prolonged growth in medium totally lacking thiamine since the UV2 strain would not be able to grow. The results are displayed in Table 3.4.

It was found that after growth in a low concentration of thiamine, (3-galactosidase

expression from all of the thiamine promoters tested, was much reduced in the UV2 strain compared with the wild-type strain. This implied that the wild-type UV2 gene product does indeed have a positive regulatory function in the expression of these thiamine genes and possibly all thiamine regulated genes. These results were preliminary; a more comprehensive study of the gene expression of thiamine genes in a uv2 background has been carried and is described elsewhere (Richards, 1996).

Numerous attempts to isolate complementing clones of uv2 and uv3 from a centromeric based yeast genomic plasmid library were made. These were all unsuccessful. However complementing clones have since been successfully isolated from a high copy YEP 13 based library. The complementing gene for both uv2 and uv3 was shown to be PDC2, a gene already implicated in the transcriptional activation of the pyruvate

49

Table 3.4

Expression from thiamine gene promoters in UV2 and W303 strains.

W303 UV2

Promoter Construct 0.02jiM thi 2.0JJ.M thi 0.02|iM thi 2.0pM thi

TH14 pUP35 1228 0 2.2 ± 3.0 0.6 ± 0.8

THI5 pRB3 261 4.8 2.9 ± 1.9 2.5 ± 0.1

THU 2 pRB4 347 3.4 2.5 ± 1.7 2.3 ± 2.3

THI6 pLK2 116 2.0 0.2 ± 0.2 0.8 ±0.1

Assays of (3-galactosidase expression from various thiamine promoter-LacZ constructs,

in a wild-type and uv2 background. Activities were measured after growth in low (0.02|iM) and high (2.0jllM) thiamine concentrations.

The results of assays carried out in the UV2 strain were performed in duplicate; the mean readings and standard errors are shown.

decarboxylase genes - PDC1, and PDC5. The details of this work and the implications of the results have been discussed previously (Richards, 1996).

3.7 DiscussionFour thiamine auxotrophic mutants have been isolated using UV mutagenesis and

171 induced by Ty insertion mutagenesis. Classical genetic analysis revealed that all the mutations were recessive to wild-type. Tetrad analysis was carried out on thirty two of these mutants, 3 UV and 29 Ty insertion mutants; they all displayed 2:2 segregation of the thiamine auxotrophy:prototrophy, indicating single locus mutations.

Suprisingly, 141 auxotrophs were found to be allelic with thi4 and although some of these were probably duplicates of the same mutational event, a minimum of four of the thi4- ty l mutations had arisen independently. It is interesting that none of the UV induced mutations were found to carry defects in the TH14 gene. This suggests that the THI4 locus could be a hotspot for Ty insertion. It is known that insertion of Tyl elements into the genome during transposition is non-random and that it is more common for the elements to insert at the 5' end of a gene than at the 3' end (Natsoulis et a l, 1989). It might therefore be interesting to see if the insertions have occurred predominantly in the 5' end of the gene rather than the 3' end; however this is not of primary concern in this work. Also it has been shown that Ty elements may be targetted to tRNA genes. This is discussed more fully in Chapter 7.

Apart from this large complementation group of thi4 mutants no other mutants were found to be defective in thiazole biosynthetic functions. This may indicate that there are no other thiamine specific genes acting in this branch of the pathway or at least none that could be identified using the screens employed here. However this is not conclusive since the auxotrophs isolated do not saturate the pathway - some of the complementation groups are represented by only one mutant.

Thirty Ty insertion mutants and three UV (UV2-4) mutants were found to complement the thi4::URA3 strain and were therefore thought to be defective in thiamine genes other than TH14. The mutant strains defined by Tyl-3 (a complementation group with 27 members), ty3-l and uv4 were believed to define novel genes which act after formation of the precursors. The gene products are perhaps involved in the phosphorylation of HMP-P or in the dephosphorylation of thiamine monophosphate. However, a strain with a mutation in a positive regulatory gene could give the same phenotype as these mutants i.e. it would be a thiamine auxotroph whose growth could not be rescued by the addition of HET or HMP to the medium. The gene products of THI2 and THI3 are thought to be positively acting regulatory elements and since thi2 and thi3 strains are auxotrophic for thiamine, tyl-3. ty3-l or uv4 could potentially bear mutations in the 777/2 or THI3 gene. However, all three strains been shown to complement both the thi2 (058-M5) and thi3 (T49-2D) mutant strains. Also

50

T49-2D displayed a different growth phenotype, being able to grow in the absence of thiamine when HET and HMP were provided in the medium together. Thus ty l-3, ty3-l and uv4 may define genes encoding novel regulatory elements and / or structural downstream enzymes.

Four thiamine auxotrophs Y02587 (thil), UV3, Tyl-128 and Ty3-7 were thought to carry mutations in genes responsible for the synthesis of HMP-PP and were believed to be non-allelic with thiS. This indicates that potentially five genes may function in the biosynthesis of the pyrimidine precursor.

The gene mutated in the strains UV2 and UV3 has been shown elsewhere to be the positive regulatory gene, PDC2 (Richards, 1996). It has been shown that although the UV2 and UV3 strains had different growth phenotypes, they did not in fact complement each other. This would be expected if they have mutations in the same gene. The patchy growth which I interpreted to represent complementation between the mutant strains may have been due to reversion, a rare mitotic recombinational event between the two mutant alleles producing a wild-type allele or intragenic complementation. However the two haploid strains did not give patchy growth on thiamine deficient medium, implying that neither mutation has a high reversion frequency in a haploid strain; but since reversion in UV2/UV2 and UV3/UV3 diploids was not tested this is not conclusive. The PDC2 gene product has already been implicated in positive activation of the structural genes of the pyruvate decarboxylase enzyme: PDC1 and PDC5 (Hohmann, 1993). That the two UV induced mutants, particularly UV2, grew poorly can now be explained since all analyses were carried out on media containing glucose; pdc2 strains do not grow well on this carbon source. The strains are unable to ferment glucose since they have no pyruvate decarboxylase activity and the alternative pathway for pyruvate metabolism, to acetyl CoA catalysed by pyruvate dehydrogenase and subsequent entry into the citric acid cycle, is repressed by glucose. The severe growth defect of the other UV induced mutant, UV1, may have similarly arisen through glucose repression.

Mutants isolated here therefore defined seven complementation groups - pdc2 (uv2 / uv3), uv4, thi4-tyl, tyl-3, tyl-128 , ty3-l and ty3-7. None of these were found to be allelic with thil or thi2; however the tyl-128 mutation was later found to be allelic with the known gene THI3. Whether the mutations were allelic with the thi80 or pho3 has not been investigated, but is unlikely as mutations in these genes do not produce thiamine auxotrophic phenotypes. The mutations were also thought to be distinct from the THIS gene family. Therefore eleven functions may be coded for by genes involved in the biosynthesis of vitamin B 1, including the positive regulatory gene PDC2 and potentially four novel thiamine genes defined by mutations isolated in this study.

51

CHAPTER FOUR

Molecular cloning of the THI6 gene.

4.1 IntroductionTwo thiamine auxotrophic strains, Ty3-1 and UV4, had been isolated whose growth

defects were not restored by supplementation with either or both of the thiamine precursors, HMP and HET. These mutants had been found to complement each other and all other known thiamine mutations that were available in the laboratory - thil, thi2 and thi4 (Chapter 3); thus they appeared to define two novel thiamine genes that function downstream of the precursors. My aim was therefore to isolate the wild-type gene(s) defined by the uv4 and ty3-l mutations, through functional complementation and to identify the function(s) for which they were defective.

4.2 Molecular cloning of the wild-type UV4 and TY3-1 genes.The simplest way to identify and clone the wild-type allele of a recessive mutation is

by use of a genomic DNA library to effect "rescue" of the mutant phenotype. Transformant clones showing the wild-type phenotype should contain plasmids that carry the wild-type allele. Therefore a yeast genomic library based on the centromeric vector, YCp50, was used to complement the mutants UV4 and Ty3-1 (Rose et al., 1987). Transformation was carried out using the high efficiency lithium acetate method and uracil prototrophic transformants were selected on SD medium supplemented with appropriate amino acids and bases: tryptophan and leucine in the case of Ty3-1; tryptophan, histidine, leucine and adenine in the case of UV4. The transformation efficiencies for each strain were: 4 x 104/pg of DNA for Ty3-1 and 4 x 103/jig of DNA for UV4. Colonies transformed to thiamine prototrophy were

selected by replica-plating onto thiamine deficient minimal medium. The number of transformants required to ensure a single copy gene would be isolated with 95% certainty, was calcuated using the following equation:

N = l nt l -P)In (1 - a/b)

where N = number of clones P = probability a = average size of clones b= total size of genome

52

Therefore since the genomic DNA inserts in the library were assumed to be 10-15kb (Rose et al., 1987) and the yeast genome is 15mb in size, the number of transformants required was calculated as between:

N = In n -0.95) In (1- 15/15,000) and

N = In H - 0.95") In (1- 10/ 15,000)

= 2994 and 4492

For the ty3-l strain, 1.75 x 105 transformants were obtained; for the uv4 strain, 4 x 1 0 3 transformants were obtained; thus for each strain the entire genome should be represented at least once. In fact 564 thiamine prototrophic transformants were isolated for the ty3-l mutation and 64 for the uv4 mutation. These figures represented higher efficiencies than expected. This may be explained by repeated amplifications of the library in E. coli resulting in a bias of the clones propagated, due to either selection being exerted for clones with smaller inserts as these elicit less of a metabolic load or, possibly, against clones carrying DNA fragments which have "deleterious" effects on the bacterium. On the other hand it could be the case that not all of these thiamine prototrophic phenotypes had been elicited by the encumbent library clones; perhaps reversion or suppression had occurred. However since the control transformations with no DNA produced no thiamine prototrophs then reversion can be ruled out and a low-copy plasmid is unlikely to exert suppression.

4.2.1 Confirmation that the library plasmids carry the complementing genes.That the thiamine prototrophies of the transformants were plasmid-derived was tested

in two ways: by curing the strains of the library plasmids and testing for reversion to thiamine auxotrophy; and by isolating the plasmids, retransforming back into the original mutants and looking for 1 0 0 % thiamine prototrophy amongst the transformants.

Selection for Ura" plasmid-free segregants was achieved by growth on 5-FOA. The Ura- 5-FOA resistant colonies that had lost the plasmid were plated onto medium lacking thiamine. One hundred of the Ty3-1 Thi+ transformants were analysed in this way; all displayed thiamine auxotrophy. Of the 64 UV4 Thi+ transformants tested only seven were found to be auxotrophic for thiamine following loss of the plasmid. It could be that the other fifty seven colonies were revertants but, as explained above, this is unlikely. This result may indicate that not all 64 strains were genuine thiamine prototrophs, indicating a leakiness or "incomplete phenotype".

Complementing library plasmids were recovered from seven of the Thi+ transformants for both Ty3-1 and UV4, transformed into E. coli and small scale plasmid preparations were carried out for each. The resulting DNAs were digested with £coRI and Sail to confirm the presence of the YCp50 7.33kb EcoRl / Sail backbone. This fragment

53

was seen in every case, as expected, indicating that the plasmids were bona fide library plasmids. Large scale preparations of these plasmids were carried out and the purified library plasmids were retransformed back into the original mutant strains using the "one step" yeast transformation method. The resultant transformants were tested for growth in the absence of thiamine; in each case 1 0 0 % of the transformants were found to be prototrophic for thiamine, confirming that all the rescued library plasmids, seven from each strain, did indeed carry the complementing gene(s).

4.2.2 Characterisation of the library clones.Restriction enzyme mapping revealed that two species of plasmid had been isolated

from each mutant. Suprisingly, the two complementing plasmids for the two mutants appeared to have identical restriction maps. It therefore seemed that two mutants which complemented each other were rescued by the same plasmids. This was confirmed by transforming the plasmids rescued from Ty3-1 into the uv4 mutant and the plasmids rescued from UV4 into the ty3-l mutant. The growth phenotypes of resulting transformants were tested. In every case a thiamine prototrophic phenotype was observed, confirming that the uv4 and ty3-J mutations were rescued by the same two clones. These clones were designated pKB15a and pKB15b; some detailed restriction enzyme mapping of the plasmids was carried out in order to define the regions within the genomic inserts where the gene(s) was (were) located. A common fragment of ~7.0kb was identified in the two plasmids (Figure 4.1). The complementing gene(s) of the ty3-l and uv4 mutations must therefore be contained within this DNA fragment. Whether the two mutants were rescued by the same gene or adjacent genes contained within the 7kb fragment of DNA had not yet been established, however the former seemed more likely. Therefore since functional complemention had been observed between the Ty3-1 and UV4 strains, it seemed possible that this might have been due to intragenic complementation; perhaps the gene encodes an enzyme with bifunctional activities which act after formation of the precursors.

4.3 Allelism of uv4 and ty3 -l with th i6Previously, a mutant with resistance to 2-aminohydroxyethyl thiazole (an anti­

metabolite of HET) had been isolated; this had been found to have reduced HET kinase and TMP-PPase activities (Kawasaki, 1993). Genetic analysis revealed that the decrease in these two enzyme activities was the result of a mutation in a single gene. thi6. Subsequently, the two enzyme activities were copurified and the bifunctional enzyme was found to be an octamer of identical 60-kDa subunits. These data implicated THI6 as a gene encoding a bifunctional enzyme.

Since ty3-l and uv4 had been shown to represent two different complementation groups, were complemented by the same clones and displayed phenotypes characteristic of a defect in functions downstream of the precursors, it was possible that they were allelic with

54

pKB15a

YCp5() DNA

HindUl BamHl Sail BamHl Sail

BamHl Sail Bamlll SaB Sail BamHl H M lll

YCp50 DNA

pKB15b

Figure 4.1

Restriction maps of the compiementing plasmids of t\3-l and uv4.

Two species of complementing plasmid were isolated from ty3-l and uv4\ their restriction maps are depicted. The patterned area represents the region of similarity between the plasmids, where the complementing gene is presumably located.

the gene defined by thi6 and that the complementing library plasmids pKB15a and pKB15b carried the wild-type THI6 gene. Soon after this conclusion had been reached, in December 1994 a paper describing the isolation and characterisation of the wild-type THI6 gene was published (Nosaka et al., 1994).

4.3.1 Comparison of the restriction maps.The sequence data surrounding the THI6 ORF, YPL214c, was obtained from the

Saccharomyces Genome Database (http://genome-www.stanford.edu/Saccharomyces/); its restriction enzyme map was analysed and compared with that of the isolated library clone, pKB15b (Figure 4.2). Although there was some discrepency in the estimated fragment sizes, the pattern of restriction enzyme sites was the same, suggesting that the gene cloned in this study was indeed THI6. This was verified in two ways - through PCR and Southern blot analyses.

4.3.2 PCR analysis.Using the published sequence primers complementary to nucleotide positions -91 to

-74 and +474 to +457 within the TH16 gene were designed. Amplification of this fragment was achieved through PCR, using as templates: the library clone, pKB15b; genomic DNA from the wild-type strain, W303; genomic DNA from the mutant, Ty3-1; and genomic DNA from the mutant, UV4. After separation of the products on an agarose gel following electrophoresis, bands were seen from each reaction corresponding to fragments of the expected size, 565bp (data not shown). This result confirmed that the plasmid pKB15b did indeed carry the THI6 gene, since it is extremely unlikely that the primers would have been able to bind to the plasmid and give an amplified product of the correct size, if the THI6 ORF was not present on the plasmid. That the product amplified from Ty3-1 DNA was the same size as that obtained from wild-type DNA indicated that there had been no Ty insertion within this 5' region i.e. within the region defined by nucleotides -91 to +474 of the THI6 sequence, in the Ty3-1 strain. It could also be concluded that the UV4 mutant strain had undergone no gross rearrangement of DNA at this locus, since an amplified product of 565bp was also obtained from its genomic DNA.

4.3.3 Southern blot analysis of the mutant strains compared with wild-type.Southern blot analysis was carried out not only to confirm that pKB15b carried the

THI6 gene, but also to locate the site of insertion of the Ty element within the Ty3-1 mutant strain.

Genomic DNAs from UV4, Ty3-1 and W303, and DNA of plasmid pKB15b, were digested with BamHl (Figure 4.3, panel A) and Sail (panel B), separated on an agarose gel by electrophoresis and subjected to Southern blot analysis. The 565bp product following PCR of W303 genomic DNA (above) was labelled with 32p dCTP and used as a

55

Figure 4.2

Restriction maps of pKB15b and the genomic DNA at the THI6 locus.

Panel A) part of the deduced restriction enzyme map of the clone isolated and described in this report, pKB15b.

Panel B) the restriction enzyme map of the clone isolated by Nosaka et al. (1994). The open arrow labelled THI6 indicates the position and direction of the THI6 ORF.The small arrows indicate the direction of and position where the PCR primers bind. Fragments used for hybridisation probes are also depicted.

0.69 | 1.0Sail BamHl Sail

2.2BattiH I

Bglll Sail

10.173 JSail

0.4890.928 2.147 12.217

THI6

Site of insertion of the Ty element

Probe 2

BamHlL__ _ 0.662 s“.flrBamHl

n

2.809 Ba%Hl__r —BgtllU------------ 2.320 Ban!m

Figure 4.3

Southern blots analysis of the THI6 locus in different strains.

Lane 1) UV4.Lane 2) Ty3-1.Lane 3) W303Lane 4) pKB15b plasmid DNA

Genomic DNA was isolated from the above strains and was digested with restriction endonucleases:A) BamHlB) SailC) BglH and BamHl.

Hybridisation probe used in Panels A and B was the 565bp PCR fragment, in Panel C the 2.8kb BamHl DNA fragment of THI6 was used.

hybridisation probe (probe 1 - Figure 4.2). A band on the autoradiograph corresponding to a fragment of approximately 2.8kb can be seen (Figure 4.3A, lanes 1 & 3, respectively), indicating that the labelled fragment hybridised to the 2.809 kb BamHl fragment in the UV4 and W303 genomic DNA. However, in the genomic DNA from strain Ty3-1 digested with BamHl a band can be seen corresponding to a fragment of approximately 9.0kb (Figure4.3 A, lane 2). This represents an increase in fragment size of around 6.2kb; the size of a Tyl element is approximately 6.0kb. Thus it was concluded that a Ty element had inserted into the THI6 gene, within this 2.8kb BamHl fragment. A band of approximately 2.8kb was also seen on the autoradiograph within the BamHl digested pKB15b plasmid DNA (Panel A, lane 4), reinforcing the conclusion that the THI6 gene was located within the insert of this library clone.

When the DNAs were digested with Sail the bands produced on the autoradiograph indicated that the probe had hybridised to fragments of ~14kb in all genomic DNAs (Figure4.3 panel B, lanes 1, 2 and 3). This suggested that a Ty element had not inserted into the 14.364kb Sail fragment within the Ty3-1 genome. Thus the site of Ty insertion was within the 2.809kb BamHl fragment, but outside the 14.364kb Sail fragment, i.e. somewhere within the 662bp BamHl / Sail fragment (Figure 4.2).

4.4 Localisation of the Ty element within the Ty3-1 genome.In order to find out where the Ty element had inserted with respect to the Bglll site,

the UV4, Ty3-1, W303 and pKB15b DNAs were digested with BamHl and Bglll and probed with the 2.8kb BamHl fragment labelled with 32p dCTP (probe 2, Figure 4.2). The banding pattern on the autoradiograph indicated that a shift in size of the 0.489kb BamHl / Bglll fragment rather than the 2.320kb fragment had occurred within the Ty3-1 genome (Figure 4.3 panel C, lane 2), with respect to wild-type (panel C, lane 3). Thus the position of insertion of the Ty element within the THI6 gene of Ty3-1 was 3' to the BgUl site.

The cleavage site of the restriction endonuclease BgUl is between nucleotides 1325 and 1326 of the THI6 ORF which correspond to codon 442. Thus the Ty element is located somewhere downstream of codon 442; within the last 98 codons at the carboxy end of the THI6 gene. Hence any protein expressed from this allele would be defective only for the carboxyl terminal 98 amino acids, at most.

4.5 D iscussionGenomic library plasmids were isolated which complemented a Ty insertion

mutation, ty3-l, and a mutation induced by UV irradiation, uv4. The clones were found to be identical, even though the mutants were able to functionally complement each other. It was subsequently shown that the mutations were allelic to a gene, THI6, encoding a multi­

56

subunit bifunctional enzyme which had recently been isolated and characterised (Nosaka et al., 1994). This work showed that a 50-amino acid region from amino acids 138 to 187 was involved in the TMP-PPase activity and that another region from amino acid 370-453 was involved in the HET kinase activity; thus the domains of the two enzyme activities are located separately in the Thi6 p. Also, a thi6 mutant which was defective in HET kinase activity but which was still able to grow in the absence of thiamine, was identified; from this it was concluded that HET is not an obligatory intermediate in the de novo synthesis of thiamine and that HET kinase is involved in the salvage synthesis of HET-P from HET, which itself is formed from enzymic degradation of thiamine and HET-P, in S. cerevisiae (Nosaka et al., 1994). This is a theory that had already been put forward (Iwashima et al., 1986; Kawasaki, 1993; White and Spenser, 1982). White and Spenser postulated that HET-P may be synthesised directly from a phosphorylated sugar, since they had shown that the C5 chain of

the thiazole is derived from a 2-pentulose, probably 2-pentulose-5-phosphate, therefore negating the need for a HET kinase function. If this theory is correct it should be impossible to obtain a thiamine auxotrophic mutant which lacks HET kinase activity but retains TMP- PPase activity, through a mutation in a single gene. Therefore the theory would be challenged if Ty3-1 proved to be defective in HET kinase activity and not in TMP-PPase activity, since ty3-l is a single locus mutation which gives rise to thiamine auxotrophy.

In strain Ty3-1, the Ty element was found to be located somewhere downstream of codon 442, within the THI6 gene and as stated above, the HET kinase activity is contained within the domain between amino acids 370 and 453. Thus it is possible that the Ty element had inserted within the DNA which encodes this domain and as a result the Ty3-1 strain would be defective in HET kinase activity. Since the UV4 strain complements Ty3-1, then it must be defective in a different domain, perhaps the domain responsible for TMP-PPase activity; then, in a heterozygous diploid carrying both the uv4 and ty3-l mutations, a conformation of the Thi6 p enzyme may be achieved which allows both activities to function, through intra-allelic complementation. However it has been shown that the carboxy-terminal 70-amino acids from 470 to 540 are required for the conformation of the protein, along with a region around amino acid 106 (Nosaka et al., 1994). This suggests that if the Ty element had inserted anywhere upstream of this domain then the Thi6 p product would be rendered defective in folding and / or oligomerisation of the enzyme; this would affect both HET kinase and TMP-PPase activities. Thus in the heterozygous diploid, it may be sufficient for a functional enzyme to be produced when only half of the subunits have this domain i.e. the polypeptides produced from the uv4 allele. This would imply that in a multimer not all the subunits are required to have this COOH terminal region for the Thi6 p to fold and have an active conformation.

57

CHAPTER FIVE

Characterisation and genetic analysis of the th il mutation

5.1 IntroductionThe first documented thiamine auxotrophic yeast strain was isolated in 1960 by

Hawthorne and Mortimer and the mutation was designated thil (Hawthorne and Mortimer, 1960). Apart from its nutritional requirements and localisation to the right arm of chromosome XIII, no other information was available regarding the strain at the beginning of this work. Preliminary analysis of the mutant strain had revealed that the mutation was not allelic with any known thiamine mutations or with any of the mutants isolated in this project and that the thiamine auxotrophy was relieved by the addition of HMP to the medium (see Chapter 3). It was therefore important to isolate the TH11 wild-type gene and identify its function within the biosynthetic pathway.

5.2 Genetic analysis of th i l5.2.1 Dominance / recessiveness test and complementation analysis

To check dominance or recessiveness of the mutation, the heterozygous diploid made by crossing KBY6 {thil) with W303a.pUP35 was tested for thiamine requirement. The strain was found to be a thiamine prototroph; therefore thil was recessive.

The heterozygous diploid was sporulated and the meiotic products analysed. In twelve tetrads analysed, the Thi“ and Thi+ alleles showed 2:2 segregation. To confirm normal segregation at meiosis the behaviour of pairs of alleles at other loci was assayed; it was found that pairs of wild-type and mutant alleles of HIS3, ADE2 and LEU2 as well as MAT a and a , all segregated 2:2. These results indicated normal disjunction and confirm that

thil is a single locus mutation.

5.2.2 Cloning the wild-type TH I1 geneAs in chapter four the YCp50-based yeast genomic library was used to isolate

complementing clones of the th il mutation. Strain KBY6 was transformed to uracil prototrophy using lpg of library DNA; transformants were obtained at a frequency of 6xl03

per |ig of DNA. Of the Ura+ transformants recovered nineteen proved to be thiamine

prototophs. In order to confirm that the Thi+ phenotype was not caused by a simultaneous chromosomal reversion event, ura3 segregants that had lost the transforming library clones were selected on 5-FOA medium and their thiamine phenotypes analysed. Plasmid-free derivatives of all nineteen strains displayed thiamine auxotrophy, indicating that their prototrophic phentoypes were not due to chromosomal events.

58

In order to confirm that the Thi+ phenotypes were conferred by the YCp50 library clones, the plasmids were rescued from six of the transformants; only two of these gave ampicillin resistant transformants of E. coli. These two clones, designated pKB19(l) and pKB19(8) were purified and the DNAs digested with the restriction enzymes HindTH and Sail for analysis by agarose gel electrophoresis. Both recombinant plasmids contained the expected YCp50 backbone of 7.33kb, and four other fragments which were identical in size, indicating that the two constructs contained the same genomic DNA insert. The two library clones were transformed back into the strain KBY6 and, in each case, 100% of the Ura+ transformants were found to be thiamine prototrophic, confirming that the genomic clones carry the thil -complementing DNA.

A restriction map of pKB19 was created and subcloning of the insert DNA revealed that a 5.185kb HindUl - Xhol fragment (pKB19c) was able to complement the thil mutation (Figure 5.1). Using a "clockwise" oligonucleotide primer homologous to vector DNA flanking the BamHl site, the cloning site of the YCp50 library vectors, nucleotide sequence data from the end of the insert DNA were obtained. These data were compared with the EMBL genomic database, using the FASTA program (Pearson & Lipman, 1988) and showed 98.6% identity, over a 382bp region, with a gene on the right arm of yeast chromosome XIII, ILV2 (YMR108w). The arrangement of restriction enzyme sites within this region of the genome was compared with the restriction map of pKB19; they were found to be very similar (Figure 5.2). Analysis of the sequence of the 5.185kb Hindlll - Xhol DNA fragment using the program Gene Jockey II (Biosoft), revealed four putative ORFs.

Further dissection of the DNA fragment carried in pKB 19c coupled with phenotypic analysis revealed that no further subclones were able to rescue the thiamine auxotrophy of thil (Figure 5.1); this included the 2.0kb EcoRI fragment (pKB19g) and the corresponding pKB19c plasmid with this fragment deleted (pKB19f). These data suggested that the ORF responsible for thil complementation spanned one or both of these EcoRI sites and thus ruled out ORFs 1 and 2 as candidates for the THI1 gene, as both are contained entirely within the fragment. Also ORF 3 was eliminated because it was contained entirely outside this region. The only ORF which spanned one or both of the EcoRI sites was YMR108w, the ILV2 gene.

5.2.3 Allelism of th i l and ilv2The ILV2 gene encodes the enzyme aceto-hydroxy acid synthase (AHAS) which

catalyses the first step in the parallel syntheses of isoleucine and valine - combining, with pyruvate respectively, a-ketobutyrate to form a-acetohydroxybutyrate, and pyruvate to form

acetolactate (Falco et al., 1985). Mutants of the ILV2 locus are obligate auxotrophs for the amino-acids isoleucine and valine (Holmberg & Petersen, 1988). However the thil strains, Y02587 and KBY6 , were found not to require isoleucine and valine for growth. An ilv2 mutant strain, STX573-3C was then obtained from the Yeast Genetics Stock Centre, UC

59

Figure 5.1

Characterisation of the THI1 clone PKB19.

Panel A) The open reading frames revealed within the sequence of pKB 19 are represented by arrows.

Panel B) Subcloning of pKB19.Fragments were isolated from the plasmid pKB19 and ligated into the YCp50 vector. Each construct was transformed into KBY6 and their

ability to rescue the Thi* phenotype was examined. Lines represent the DNA fragments carried in the various subclones.

HimWWXbaX EcolW Xbal EcoRI AViol Xbal Sail

J dORl’l . -ORF3

0RF2%

ILV2

I------1

i lkb i

Construct:

pKB19a

pKB19b

pKB19c

pKB19d

pKB19e

pKB19f

pKB19g

pKB19h

Rescue thil?

+

+

3.25kbHind III

H 5.0kbXhol SailH l.Okb I 2.02kb

3.0kbEcoRI

< - 2.539kb

Hindlll EcoRI0.515 2.024kb

>

EcoRI

J 0.953kb | Xhol Sa il

5.185kb

ORF I ^ORF3

ORF2lkb

ILV2

Figure 5.2

Comparison of restriction maps.

Panel A shows the map of pKB19, elucidated from restriction mapping.Panel B shows the restriction map predicted from the ILV2 sequence data (Falco et al., 1985). The ORFs encoded by the DNA are also shown.

M W W H W

PlasmidDNA

Berkeley and tested for ability to grow in the absence of thiamine; it showed no requirement for thiamine. Interestingly, the thil complementing clone, pKB19c, conferred isoleucine and valine prototrophy when transformed into this strain, indicating that it carried a bona fida ILV2 allele. In order to perform the reciprocal complementation experiment a bona fide ILV2 wild-type clone (pE16/8-C4) was obtained from Professor M. Keillard Brandt (Copenhagen). When this plasmid was transformed into strain KBY6 it was able to restore thiamine prototrophy. This revealed an intriguing paradox: the thil mutant was not isoleucine and valine requiring and an ilv2 mutant was not thiamine requiring, yet both mutations were complemented by the same fragment of DNA. One explanation could be that YMR108w is the wild-type locus for one of the mutations and a suppressor of the other. However the complementing fragments were carried on low copy vectors, which make suppression unlikely. It therefore became important to disrupt this locus and examine the phenotype of the resultant mutants. Additionally, examination of the meiotic segregation of the ilv2 and thil mutations would resolve whether they were allelic and clarify the paradox.

5.2.4 Disruption of the ILV2 locusIn order to make a deletion strain of the ILV2 locus the 'one-step' gene replacement

method was employed. The strategy for constructing the ILV2 disruption cassette is shown schematically in Figure 5.3. The plasmid pKB19c was digested with the restriction endonuclease Xbal and the large fragment religated to itself thereby deleting 1.35kb of the gene, approximately two thirds of the coding region, from nucleotide 769 to 2121. However to do this it was first necessary to pass the plasmid DNA through a dam~ strain of E. coli, since one of the Xbal recognition sequences (5'-TCTAGA-3') was overlapped by the Dam methylation site (5'-GATC-3'). Methylation of the A residue prevented cleavage by the Xbal endonuclease. Having obtained unmethylated plasmid DNA, it was then possible for the 1.352kb Xbal fragment to be excised and the vector religated to itself, creating the construct pKB19h. This plasmid was transformed into KBY6 and as expected was unable to rescue the thil auxotrophy.

A DNA fragment carrying the URA3 gene was released from pKB22a by Xbal digestion and ligated into XM -cut pKB19h to give vector pKB22, carrying the ilv2::URA3 construct. This plasmid was then digested with Hindlll and £coRI, the fragments separated by agarose gel electrophoresis so that the 2.29kb was isolated away from the YCp50 backbone, as this also carries a copy of the URA3 gene. Thus the disruption cassette was released and was transformed into the ILV2 yeast strains W303a and W 303a.

Approximately 50 Ura+ transformants were obtained for each strain. Twelve transformants of each type were streaked to single colonies and replica-plated onto minimal medium to test their phenotypes with respect to isoleucine & valine (IIv) and thiamine. Only one of the 24 transformants tested was IIv+ whilst all 24 strains were Thi+ ! Thus it appeared that 23 out of

60

Figure 53

Disruption of the ILV2 (YMR108w) locus.

The plasmid pKB19h was digested with Xbal and ligated with URA3 fragment released from the pUC19 - based plasmid, pKB22a by Xbal digestion. The ilv2::URA3 disruption cassette was released by HindHl / £coRI digestion and was transformed into the ILV2 strain W303.

24 transformants had been disrupted at ILV2 (Ailv2) but none were thiamine auxotrophs,

i.e. they were phenotypic ally 77/77.The disruptions of ILV2 in the MATa and M AT a strains were confirmed by

Southern blot analysis. Genomic DNA was isolated from strains W303a, Y02587, W303aAz7v2:: URA3 and W303otAilv2:URA3 and 5jig of each DNA was digested overnight

with TscoRI; a small amount of plasmid pKB19c DNA was similarly digested The products were separated by agarose gel electrophoresis and after depurination, denaturation and neutralisation were transferred to a Hybond-N+ nylon membrane and hybridised with the 2.0kb EcoRA fragment (Figure 5.2) labelled with fluorescein (Gene Images kit). The autograph of the filter is shown in Figure 5.4. DNAs of strains W303a and Y02587 and the pKB19c plasmid DNA yielded single hybridising fragments of about 2kb, whi(ch corresponded to the 2.024kb fragment within the wild-type ILV2 gene (lanes 1, 2 and 5). However DNA of the Ura+ transformants yielded a larger single band of approximately 6.0kb. This corresponded to the size expected (5.968 kb) of a recombinant allele created by insertion of the URA3 marker fragment, as one of the EcoRI sites would be destroyed during construction. These results clearly showed that the ILV2 locus had been disrupted with URA3 in both W303a and W303a.

5.2.5 Nutritional characteristics of th il and i7v2 mutantsIt was suspected that the presence of isoleucine and valine in the medium was

obscuring the requirement for thiamine in the disruptant strains, as they were able to grow on minimal medium supplemented with isoleucine and valine alone. However it was then observed that the haploid thil strains, Y02587 and KBY6 , did not in fact require thiamine for growth when isoleucine and valine were supplied in the medium. The true phenotype, therefore, of the thil mutation appeared to be one of thiamine auxotrophy in the absence of isoleucine and valine.

5.2.6 Segregation of i/v2 and th il allelesThe W303aAz7v2 strain was crossed against the thil strain KBY6 ; the heterozygous

diploid exhibited the thil phenotype i.e. it was a thiamine auxotroph in the absence of isoleucine & valine. The diploid was sporulated, ten tetrads were dissected and the meiotic products analysed for segregation of thil and ilv2 alleles. Segregation of alleles of nutritional marker genes was also assayed to verify that disjunction at meiosis had occurred normally. The wild-type versus mutant alleles at the 77753, LEU2, URA3 and ADE2 loci all segregated 2:2, confirming normal meioses. Of the ten tetrads dissected all displayed 4:0 segregation of Thi+:Thi" phenotypes in the presence of isoleucine and valine. However in the absence of isoleucine and valine only two spores from each tetrad were able to grow with thiamine supplementation; no spores were able to grow in the absence of both thiamine and isoleucine and valine. Thus, the thil phenotype could be distinguished from the IIv- phenotype since it

61

Figure 5.4

Southern blot to show disruption of ILV2.

Genomic DNAs were digested with the restriction endonuclease ZTcoRI and hybridised with a fluorescein labelled 2.0kb £cc?RI fragment from pKB19c.

Lane 1) W303 Lane 2) Y02587

Lane 3) W303aAilv2::URA3

Lane 4) W303aAilv2::URA3

Lane5)pKB19c

Below is a schematic diagram of the restriction enzyme cleavage sites within the wild-type and disrupted loci.

6.0 kb £coRI fragment

2.0 kb £c<?RI fragment

7LV2 probe2.01cb

XbalEcoRI ^

ILV2

XbalEcoRl Xbal

URA3

EcoRl

-tP - 6.0kb

could grow without isoleucine and valine when thiamine was present in the medium. Also 2 :2 segregation of ilv2 and th il alleles was demonstrated, confirming allelism of the mutations. Moreover the absence of any Non-Parental Ditypes or Tetratypes indicated that the thil and ILV2 genes are tightly linked.

These observations support the proposal that the true phenotype of the thil mutation was one of thiamine auxotrophy in the absence of isoleucine and valine.

5.3 Cloning the th i l alleleThese paradoxical observations lead to the conclusion that TH11 has no function in

the biosynthesis of thiamine. Since it was known that the ILV2 gene product, AHAS, required TPP as a coenzyme (discussed in Chapter 1), it seemed likely that thil was an allele of 1LV2 encoding a functional AHAS enzyme but with a enhanced requirement for thiamine. Such a situation could arise if the Thilp had a reduced binding affinity for TPP; thus by adding exogenous thiamine to the medium, the internal concentration of TPP would be elevated, due to active uptake and sequestration of thiamine by conversion to TPP, which is stored within the cell (Iwashima et al., 1973; Suzuoki, 1955). The wild-type AHAS must have a sufficiently high affinity for TPP for it to be bound at the endogenous level, allowing Ilv2p to be functional and producing isoleucine and valine in the absence of exogenous thiamine. In order to investigate this hypothesis further and to find supporting evidence it was necessary to clone the mutant thil allele.

Cloning was carried out using the technique known as Gap repair (see Figure 5.5). The YCp50 based plasmid pKB19h used in the disruption experiment and carrying the 5' and 3' ends of the ILV2 gene with the intervening 1.35kb deleted, was digested at the unique Xbal site and the resulting linear DNA molecules transformed into the thil strain KBY6 . Any Ura+ transformants should carry the plasmid with the gap having been repaired by homologous recombination with the chromosomal thil locus. If the plasmid had simply integrated into the chromosome then mitotic catastrophy would have ensued since the chromosome would be dicentric and would break at mitosis.

Plasmid DNAs from two different Ura+ transformants were isolated from yeast, amplified in E. coli and transformed back into the Ailv2 strain to check the phenotype of the

isolated clones. Both clones gave the thil phenotype i.e. they were auxotrophic for thiamine in the absence of isoleucine and valine, but the addition of either thiamine or isoleucine and valine to the medium, restored growth. The phenotype of W303ilv2::URA3 transformed with one of these plasmids, pKB24 is shown in Figure 5.6.

62

Cut ends of DNA stimulate recombination with homologous chromosomal locus.

Plasmid DNA is linearised

Xbal Xbal pKB19h

Homologousrecombination

iChromosomalDNA

Xbal XbalpKB24

Figure 5.5

“Gap Repair” of pKB19h.

The plasmid was digested with Xbal and transformed into the thil strain, KBY6; the gap was repaired by two homologous recombination events and the thil allele was cloned into plasmid pKB24.

I+THI AM INE +ILV

i ^ \ n KB:4 pKB 1 9 /^ 1

I Y C p 5 o \ y' Mlv2 }

1 pKB25/ \ pKB30 i

1 ^ / pKB26 nKB28\>^j

-THIAMINE -ILV

-THIAM INE +ILV +THIAMINE -ILV

Figure 5.6

Phenotypic growth studies.

Single colonies of derivatives of the W303Ai/v2 strain carrying various plasmids were streaked on minimal media, as indicated.

pKB 19 - ILV2 allele pKB24 - thil allelepKB25 - construct 1 ILV2::Xbol-Xbal thilpKB26 - construct 2 th il::Xbal-Xbal 1LV2pKB28 - construct 3 lLV2::Spel-Spel thilpKB30 - site directed mutagenesis allele YCp50 - empty vector

Ailv2 - no plasmid

5.4 Acetolactate synthetase assaysIn order to test the hypothesis, it was essential to establish whether the AHAS

activity of the thil strain had an altered TPP dependency. Therefore acetolactate synthetase (ALS) activity, one of the two activities of AHAS, was measured in Ailv2 strains carrying

various alleles on low copy plasmids: pKB19 (ILV2), pKB24 (ilv2-thil) and YCp50; the results are displayed in Figure 5.7, Panel A.

Permeabilised cells carrying the ILV2 allele i.e. with wild-type ALS enzyme, were found to have high levels of activity that showed little dependence on TPP concentration over the range 50 - 200|J.M; even with no additional TPP high levels of acetolactate were

produced, indicating that the extracts contained sufficient endogenous TPP for almost full ALS activity. In contrast, extracts of cells carrying the ilv2-thil allele (pKB24) showed no ALS activity in the absence of exogenous TPP; addition of the cofactor stimulated activity but never to the very high levels recorded in extracts of ILV2 cells. Thus these cells may possess a mutant form of the enzyme with reduced activity and altered TPP dependency. Note that a very low level of TPP dependent "ALS" activity was measured in extracts made from cells deleted of the ILV2 gene (z/v2A): since ILV2 is a single copy gene the activity

must be due to other enzyme(s) that can carry out a ALS-type reaction converting pyruvate to a-acetolactate, as measured in this assay. On the other hand these data could merely be

indicative of the experimental error of the system, since the activities of the thil and ILV2 enzymes are significantly greater than those of ilv2A.

If TPP is treated as a substrate rather than a cofactor, then a double reciprocal "Lineweaver-Burk" graph of 1/y against 1/[TPP] could be plotted. This is shown in Figure 5.7, Panel B, with the plots of ILV2 and ilv2-thil having been extrapolated so that Vmax and Km could be estimated. Note that the strict accuracy of the calculations and

extrapolations cannot be guaranteed since the activity data for the Ilv2p enzyme was close to Vmax at all the TPP concentrations i.e. the TPP levels were almost saturating. However it

was possible to compare the kinetics of the 7LV2-derived enzyme with those of the enzyme from the of ilv2-thil allele. Thus to the first approximation extrapolation of the data gave Vmax and Km values for the wild-type enzyme (that expressed from the ILV2 allele) of 2000 units and 10|iM. In contrast the Vmax and Km values for the mutant, th il-derived, ALS enzyme were 750 units and 23p.M, respectively.

Although these data may not be strictly accurate, it can be seen that the Vmax of wild-

type Ilv2p enzyme was greater than that of the mutant form (ilv2-thilp), which indicated a faster rate of substrate turnover for a given concentration of TPP. Also the Km of the Ilv2p

enzyme was smaller than that of the ilv2-thilp enzyme indicating stronger binding of TPP to Ilv2p than to the ilv2-thilp. This is consistent with the hypothesis.

63

Figure 5.7

ALS activities versus TPP concentration.

Panel A) ALS activities of permeabilised extracts of cells carrying various ilv2 alleles. Activity is measured as OD5 4 0 nm / mg protein / min, calculated as total

acetoin + acetolactate - background acetoin (see Materials and Methods).Each reading is the mean of two independent experiments carried out on the same day with the same cell extract. Error bars indicate the standard error between the two readings.

Panel B) Lineweaver-Burk plot of 1 / ALS activity versus 1/TPP concentration for ILV2 and ilv2.thi. The graph intersects the X-axis at -1 / Km and the Y-axis

at 1/Vmax.

- 0.1

- 0 .09 -

- 0 .08 -

- 0.07

^ - 0 .06 -

« - 0 .05 -

- 0 .04 -

K>i- 0.03 -

- 0 .02 -

- 0.01

1 / ALS activity x 10-3

0 .01 -

0. 02-1

ALS activity (QD5 4 0 1 HH / mg protein / min)

5.5 Mutation mapping by fragment exchangeIn order to locate the mutation(s) within the thil allele subcloning was carried out,

swapping restriction fragments of the thil gene with the wild-type ILV2. Three fragment exchange constructs were made, as depicted in Figure 5.8. Firstly the 1.35kb Xbal fragment from the thil clone, pKB24, was isolated and ligated into Xbal digested pKB19h, making pKB25. Secondly, the Xbal fragment from pKB24 was replaced with the Xbal fragment from pKB19c making pKB26. Thirdly, the SpeI fragment from pKB19e was deleted and replaced by the Spe I fragment from pKB24 making the construct pKB28. All of these plasmids were transformed into strain W303Ailv2 and their phenotypes analysed. The

growth patterns of strains on various media are shown in Figure 5.6 (pKB25, pKB26 and pKB28). The strain carrying pKB19c, the wild-type 1LV2 was able to grow on all the media i.e. it required neither thiamine nor isoleucine and valine. The strain carrying the thil clone, pKB24, was able to grow in the presence of isoleucine and valine, whether thiamine was present or not. It was also able to grow in the absence of isoleucine and valine, if thiamine was provided, but not on medium lacking thiamine, isoleucine and valine. The strain carrying pKB25 behaved like 1LV2, whereas strains carrying pKB26 and pKB28 behaved like the thil mutant. These data indicated that the thil mutation was not contained within the X bal fragment, but was located within the upstream 0.691 kb Spe I fragment. Thus the mutation appeared to be outside the region of DNA deleted for the "gap-repair" procedure.

5.6 Characterisation of the th il mutationIn order to establish exactly where the thil mutation(s) was (were) located and to

determine its (their) effect on the AHAS amino acid sequence, the nucleotide sequence of the th il allele was determined. Using the published sequence, primers were designed to the ILV2 gene at 300 to 600bp intervals along both strands of the ORF (Figure 5.9). Using the ABI cycle sequencing method, the entire length of the th il ORF was sequenced and compared with the published ILV2 sequence using the Mac Vector "Align" package. A single nucleotide base change was found within the sequence of th il. Figure 5.10 shows the electropherograms of the sequenced data surrounding the mutation in both thil and ILV2

The mutation, located to nucleotide position 528, creates the transversion:

T5 2 8 — ^ ^528 bringing about the codon change:GAT — ^ GAG which creates the conserved aa substitution

Asp 1 7 6 Glui76

This mutation also creates a new cleavage site for the restriction endonuclease Stul. 5’-ATGCCT-3' —► 5-AGGCCT-3'

64

1) pKB25

Xbal Xbal

4 th il

2) pKB26

Xbal Xbal

ILV2y r / r y r y r > r

thil

Xbal I

XfozlSpel

3) pKB28 > v V v v V V v 'y 7 r T ? ^ V v V f/rf7

.Spel

- L

Growth of A i7v2 strains carrying various plasmids:

Plasmid +thi +ilv +thi -ilv -thi +ilv -thi -ilv

pKB19 (ILV2) + + + +pKB24 (thil) + + + -

pKB25 (1) + + + +

pKB26 (2) + + + -

pKB28 (3) + + + -

Figure 5.8

Reciprocal exchange of plasmid fragments.

The Xbal fragment from pKB24 (thil) was ligated into the Xbal site of pKB19h (ILV2 clone with Xbal fragment deleted), creating construct 1 (pKB25).The Xbal fragment from pKB19c was ligated into the Xbal site of pKB24h (thil clone with Xbal fragment deleted), creating, construct 2 (pKB26).The Spe I fragment from pKB24 (thil) was ligated into the Spe I site of pKB18h (ILV2 clone with Spe I fragment deleted), creating construct 3 (pKB28).

2067bp528bp

Stul

Spe I Spe I Xbal Xbal

lkb

KB 16 ^

KB 15 r

KB18^

KB 10

KB 11

KB 12

^ KB13

KB 14

Figure 5.9

Sequencing analysis of the th il gene.

Arrows indicate the direction of each primer used and the extent of the sequence data obtained from it. The asterix indicates the site of the base change.Specific sites where the primers bind are given in Materials and Methods.

Figure 5.10

Electrophoregrams of sequence data from the th il and ILV2 genes.

Plasmids carrying the ILV2 (pKB19c) and thil (pKB24) alleles were used in the sequencing analysis.Shown opposite are sequence data from the region around the mutation (nt 518 to 538) from:Panel A) coding strand of the ILV2 gene Panel B) coding strand of the thil gene Panel C) non-coding strand of the thil gene

Oligonucleotide primer KB 16 was used for the coding strand sequence and KB 14 for the non-coding strand. Specific sites where the primers bind are given in Materials and Methods.

o

<

u

o

V

u

o

f — I

<

o<u

o

o

<

<

u

u

The base change was observed in four different sequencing reactions, two on each strand of the gene. The sequencing data revealed that the thil mutation was located lOObp upstream from the 5' X ba l site used for gap repair cloning of the th il allele. Thus recombination between the ILV2 and thil sequences must have extended well beyond the ends of the gapped, linearised plasmid DNA.

5.7 Site-directed m utagenesisTo confirm that the single base change, T5 2 8 —^ G528> was solely responsible

for conferring the th il phenotype, a PCR-based site-directed mutagenesis strategy was employed. The schematic outline is shown in Figure 5.11.

Oligonucleotide primers incorporating the altered base, were designed complementary to both strands of DNA incorporating the 25bp region surrounding the observed mutation - KB20 and KB 19. These primers along with KB21 and KB 14, which annealed to the flanking ILV2 DNA upstream and downstream of the mutation site, were used to amplify the two halves of the ILV2 gene by PCR. The products of these two reactions were recovered, mixed together, denatured and used as template for PCR using only two flanking primers KB21 and KB 14. The product obtained was a 1.24kb fragment of 1LV2 sequence with the introduced mutation. This was purified and digested with Spe I, to release the 691 bp fragment, containing the site-directed mutation. This fragment was ligated into Spe I cut pKB18h to make pKB29, which contained the mutated ILV2 gene on pUC19 vector. Three separate site-directed mutagenesis clones were created in this way and each was digested with the restriction endonuclease Stul, to confirm that the mutation had been correctly incorporated into each one. All three had a Stul site at the expected position. The 691bp Spe I fragment of each construct was sequenced, using primers (KB 14, KB 16 and KB21, described in Materials and Methods). Two of the constructs had an additional mutation at nucleotide 440 an A —» G transversion, creating the codon change GAA —> GGA and making the non-conservative amino-acid substitution, Glutamate—» Glycine. These two

constructs were therefore discarded from further analyses. The third construct, however, contained no other mutations and as confirmed by the Stul digest, had the site-directed mutation incorporated. Therefore the mutated ILV2 gene was released on a Hindlll - Sail fragment and ligated into H indlll - Sail digested YCp50, creating the site-directed mutagenesis clone, pKB30. When transformed into the W303Ailv2 strain this plasmid

conferred the thil phenotype i.e. it was able to grow in -Thi+Ilv and +Thi-Ilv media but not in -Thi-Ilv medium, confirming that the single base change was solely responsible for the altered th il phenotype (Figure 5.6, pKB30).

65

Figure 5.11

Schematic plan for PCR based site-directed mutagenesis.

PCR 1 and 2 reactions involved amplifying two fragments o f the ILV 2 gene, incorporating the designed mutation. The products were recovered, mixed together, denatured and used as template for PCR 3. Two flanking primers, KB21 and KB 14 were then used to amplify 1.24kb of the ILV2 gene surrounding the introduced mutation. The product was purified and digested with the restriction endonuclease, Spe I, to release the 691 bp mutated ILV2 fragment which was ligated into Spe I cut pKB18h to make pKB29.Arrows indicate site and direction where the primers bind.

Base change200bp

0 2 1 --------------------* KB20

D 1 9 J *---------* K B !4

KB21 =±________ :■---------- S=KB14

I

1Cleave with Spe I

691bp fragmentSpeI \SpeI

ILigate into Spe I cut pKB18h

ISite-directed mutagenesis clone, pKB29

PCR1

PCR2

PCR3

5.8 Confirmation of phenotype for the site directed mutantIn order to verify whether the thil growth phenotype of an Ailv2 strain carrying the

pKB30 plasmid was reflected in enzymatic activity, ALS activty assays were carried out on permeabilised cells of the strain, as described in Section 5.4. The data obtained from the enzyme produced by the site-directed mutagenesis clone, pKB30, were virtually identical to those obtained from the original thil mutant allele (Figure 5.12). There was very low ALS activity when no TPP was added to the enzyme assay mixture compared to the wild-type Ilv2p enzyme which gave high levels. Increasing ALS activity was produced from the Ilv2p- sdm enzyme with higher TPP levels, consistent with Ilv2-thilp.

These data suggest that the single base change observed in the sequence of thil was solely responsible for a reduced TPP-binding affinity of the ALS enzyme product, which manifested itself in a growth requirement for exogenous thiamine.

5.9 Confirmation of the S tu l site in the genome of the th i l strainThe final confirmation that the thil mutant strain carried a substitution within the

1LV2 ORF was possible since this base change created an additional site for the restrictionendonuclease Stul. Therefore genomic DNAs were isolated from the thil strain KBY6 andtwo "wild-type” strains, DG622 and W303, as the parent strain for the original thil strain(Y02587) was not available. 5jig of each DNA was digested overnight with Stul. The

products were separated by agarose gel electrophoresis and subjected to Southern Blotanalysis. The transferred DNA fragments on a nylon membrane were then hybridised withthe 2.0kb EcoRl fragment (Figure 5.2) labelled with fluorescein. The autograph of the filteris shown in Figure 5.13. DNAs of strains W303a and DG622 yielded single hybridisingfragments of approximately 23kb (lanes 1 and 3), which must correspond to the 23.279kbStul fragment which encompasses the ILV2 locus. However DNA of the KBY6 strainyielded two smaller bands of approximately 5kb and 19kb, which correspond to the sizesexpected (4.563 and 18.716kb) if an extra Stul restriction site had been created at nucleotide528 of the ILV2 ORF. These results clearly showed that a mutation was present in theoriginal thil allele which led to this additional Stul restriction site.©

66

2250-

ILV2

.sI 1500-

a1* 750 -

11

2

i/v2A

200150100TPP concentrations (|iM)

Figure 5.12

ALS activity of the product of the ilv2-sdm allele.

ALS activities of permeabilised extracts of cells canying various ilv2 alleles. Activities measured as described in the legend of Figure 5.7. Each reading is the mean of two independent experiments carried out on the same day with the same cell extract. Error bars indicate the standard error between the two readings.

< - l lk b

<------ ~5kb

18.716kb 4.563kb ^ 6.331kb»|

ILV2 {thil

Stul EcoKL (Stul) EcoKL Stul Stul

Probe

Figure 5.13

Southern blot of wild-tvpe and KBY6 genomic DNAs.

Genomic DNAs were digested with the restriction endonuclease Stul and hybridised with a fluorescein labelled 2.0kb FcoRI fragment from pKB19c.

Lane 1) W303 Lane 2) KBY6 Lane 3) DG622

Below is a schematic diagram of the restriction enzyme cleavage sites at the ILV2 (thil) locus.

5.10 DiscussionThe thil gene is allelic with ILV2 the structural gene encoding the aceto hydroxy acid

synthase enzyme. Its apparent phenotype of thiamine auxotrophy is only manifest in the absence of the branched chain amino acids isoleucine and valine; it could therefore be more accurately described as a thiamine, or isoleucine and valine auxotroph i.e. either the presence of thiamine or isoleucine and valine is able to restore its growth defect, on minimal medium.

Having cloned and sequenced the th il gene the mutation causing the thiamine / isoleucine and valine auxotrophy phenotype of the mutant was shown to be elicited through a single base change, which translated to a conserved amino acid substitution Asp —» Glu. The

position of the mutation was situated towards the amino terminal end of the protein, at residue 176, quite distant from the conserved TPP-binding consensus sequence which is located towards the carboxy terminus (codons 547 - 577). It was also located outside the 1.35kb Xbal fragment deleted for gap repair, lOObp upstream from the 5' Xbal restriction site; this was somewhat surprising. However the subcloning strategy which involved hybrid plasmids between the wild-type ILV2 gene and the mutant thil gene confirmed that the thil mutation lies outside this X bal fragment and within the 0.691 kb Spe 1 fragment, which is situated towards the 5' end of the gene (-114 to 577bp).

A PCR based strategy for site-directed mutagenesis was used to show conclusively that the observed single nucleotide base substitution was responsible for the mutant phenotype of th il. However in order to confirm the hypothesis for the phenotype, i.e. that the thiamine auxotrophic phenotype of thil was due to the mutant AHAS enzyme with a reduced binding affinity for TPP, biochemical evidence was required. Enzyme assays which measured the amount of acetolactate produced from pyruvate in a given time, were carried out on permeabilised cells of yeast strains carrying different alleles of ILV2. The subsequent data indicated that for any given concentration of TPP the wild-type enzyme produced more acetolactate than the enzymes encoded by the thil allele or the site-directed mutant allele. If no TPP was added to the enzyme assay mixture then the wild-type enzyme was still able to produce acetolactate at a level almost equal to the activity attained when a high level of exogenous TPP (200JJ.M) was available, implying that the endogenous level of TPP was

sufficient for the enzyme to be fully active in vivo; the two mutant enzymes however had very low levels of activity in the absence of an exogenous source of TPP. Thus the mutant enzymes required a higher level of TPP to be available for them to be functional, implying that in vivo the enzymes would only be able to produce isoleucine and valine when thiamine was added exogenously. These results support the hypothesis.

It has been indicate that the residue Asp176 is important in the binding of TPP to the enzyme AHAS and that a D176E mutation causes the affinity of the enzyme for its substrate to be reduced. Therefore Asp176 may form part of the binding site for TPP in AHAS, although this residue has not been implicated in the TPP binding site, either in the E. coli AHAS isozyme II through homology modelling with the crystallographic structure of POX

67

(Ibdah et al., 1996) or, as far as I am aware, in an equivalent position in any other TPP- dependent enzymes. Another possibility could be that the residue may not be directly involved in the binding site itself, but may affect its conformation by causing the protein to fold in such a way that if this residue was altered then the cofactor would no longer be as efficiently bound. Such a conserved amino acid substitution, however, would be unlikely to cause any gross perturbance to the tertiary folding of a protein. Only if the crystallographic structure of S. cerevisiae AHAS was resolved will the significance of the Asp176 residue, in the binding of TPP, be revealed.

68

CHAPTER 6

M olecular cloning of the THI2 and TH13 genes.

6.1 Introduction.

Two mutations known as thi2 and thi3 give rise to thiamine auxotrophic phenotypes and have been shown to affect activation of the thiamine regulated genes - PH 03, THI6, THI80, THI4, THI5 and THI12 (Burrows, 1997; Kawasaki, 1993; Nosaka et a l, 1993). Thus the Thi2p and Thi3p products are potentially important regulatory proteins and although their role(s) had previously been assumed from the effects of the mutations, their wild-type genes had not yet been characterised. Therefore my aim was to isolate the THI2 and THI3 wild-type genes in order to elucidate their exact functions in thiamine gene regulation.

Complementation and analysis of another mutation is described in this chapter, since it was found to have a defect in the THI2 gene: the complementation group represented by tyl-3 which was only able to grow in the presence of thiamine. This mutation was shown to be recessive and in a single gene (see Chapter 3). Complementation analysis had indicated that the mutation was non-allelic with thi2.

6.2 M olecular cloning of THI2,A thi2 strain bearing a ura3 mutation, KBY7, was transformed with lpg of the

YCp50 based yeast genomic library and transformants were selected on minimal medium lacking thiamine. Eight of approximately 6000 Ura+ transformants were found to be thiamine prototrophs and these all reverted to thiamine auxotrophy when the library plasmids were lost, indicating that the plasmids were conferring the thiamine prototrophic phenotypes. In order to confirm this, the plasmids were isolated from all eight Thi+ transformants and were transformed back into the thi2, ura3 strain; 100% of the Ura+ transformants displayed thiamine prototrophy. It was found through restriction analysis that all eight plasmid DNAs were identical; one of these was designated pKMl.

6.2.1 Analysis of pK M lUsing oligonucleotide primers complementary to the DNA flanking the cloning site

of the YCp50 vector, nucleotide sequence data from the end of the insert DNA were obtained. These data were compared with the EMBL genomic database, using the FASTA program (Pearson and Lipman, 1988). One end of the pKMl insert was found to lie within an ORF of unknown function on chromosome II, YBR239c. Restriction mapping of pKMl plasmid DNA revealed that the genomic insert was approximately 6.4kb. The restriction map and putative ORFs encoded by the DNA insert carried on pKMl are shown in Figure 6.1.

69

6.2.2 Identifying the TH12 ORF.In order to identify the complementing THI2 ORF within the genomic DNA insert,

pKM l was subjected to Tn1000 based bacterial transposon mutagenesis (Sedgwick and Morgan, 1994). When the resultant Tn1000 disrupted plasmids were transformed back into the thi2 mutant only one failed to rescue the thi2 mutation; this was designated TnpKMl (non-rescuer of thi2). The complementing ORF was identified by locating the site of insertion of the Tn1000 within TnpKMl. Restriction mapping revealed that the transposon had inserted within a 1.14kb EcoK l fragment. This fragment spans most of an ORF designated YBR240c and some of its promoter (Figure 6.1). Therefore it was concluded that thi2 was complemented by YBR240c.

6.3 Disruption of the YBR240c locus.In order to show that YBR240c did indeed represent the wild-type THI2 gene and

not a suppressor of the mutation, the locus was disrupted using the one-step gene disruption method. The TnpKMl plasmid was digested with Hpal and Narl to release a fragment of approximately 10.6kb which contained the entire Tn1000::URA3 sequence with flanking DNA from the YBR240c locus (Figure 6.1). The fragment was isolated from an agarose gel and transformed into a TH12 yeast strain - W 303a. Transformants were selected on SD

medium lacking uracil then four of these colonies were purified and tested for thiamine auxotrophy. All four W303thi2::Tnl000 colonies were found to be auxotrophic for thiamine, suggesting that the TH12 gene had been disrupted.

Southern blot analysis was carried out to confirm that integration of the Tnl000::URA3 fragment had occurred at the YBR240c locus. Genomic DNAs were isolated from the Thi* disruption strain W 303::Tnl000 and the parent strain W303. These were digested with Hpal, the fragments separated by agarose gel electrophoresis and transferred to a nylon membrane. A 4.7kb Hpal /N a r l fragment from the pKMl plasmid was labelled with fluorescein and used as a hybridisation probe. A single H pal fragment of size 5.5kb hybridised to the probe within the genomic DNA of W303 (Figure 6.2A, lane 2) corresponding to an undisrupted YBR240c locus. However a single -1 lkb DNA fragment hybridised to the probe within the W303Athi2 DNA, indicating that YBR240c had been

disrupted by the 5.9kb Tnl000::URA3 DNA fragment (lane 1).That the wild-type THI2 locus had been successfully disrupted was confirmed

genetically by crossing the disruption strain, W303athi2::Tnl000, with a strain carrying the

original thi2 mutation, KBY7; the resulting heterozygous diploid was found to be auxotrophic for thiamine. Since the mutations did not complement, thi2 and Y'&RlAOcv.TnlOOO are allelic. Hence YBR240c is the THI2 ORF.

70

Figure 6.1

Restriction map of pK M l.

The restriction map of the 6.4kb DNA insert of plasmid pK M l and the candidate ORFs encoded by this DNA are displayed.The site of insertion of Tn1000::U RA3 fragment, the DNA fragment used for the hybridiastion probe and the site of excision for disruption are indicated by arrows.

Fragment where TnlOOO::URA3 has inserted 4-----------►

098kb

2.19kb 1.775kb —

YBR241cYBR240cYBR239C

EcoRl EcoRl NarlNarl Hpal EcoRlHpal EcoRl

----------

5.555kb 1.147kb

4.727kb Hpal - Narl fragment used for hybridisation probe and disruption

VectorDNA

1 2

m

H

► - l lk b Hpal fragment

► ~5kb Hpal fragment

Figure 6.2

Southern blots of YBR240c disruption.

Genomic DNAs were isolated from wild-type (W303) and W 303::Tnl000 strains and digested with Hpal.

Lane 1 - W 303::Tnl000 DNA Lane 2 - W303 DNA

The 4.7kb H pal / NarI fragment from pKMl was used as a hybridisation probe.

6.4 Thi2p is a Zn-finger DNA binding proteinSince YBR240c ORF was confirmed as the THI2 gene, the nucleotide sequence was

translated, using the program Gene Jockey II (Biosoft). The deduced gene product was found to be a 450 amino acid protein. The S. cerevisiae genome sequencing project had recently been completed and as a result this ORF had already been analysed (http://speedy.mips.biochem.mpg.de/mips/yeast/). It had been identified as a probable transcription factor with a DNA binding domain, defined by a putative Zn2-Cys6 zinc-finger

motif, similar to that found in Gal4p. The amino acid sequence with the zinc-finger motif highlighted is shown in Figure 6.3.

6.5 Allelism o f th i2 and t y l - 3 ,A Ty induced thiamine auxotroph, Tyl-3, was functionally complemented by a

library plasmid designated pM Dl. Restriction analysis of this plasmid revealed that it was very similar to the thi2 complementing clone, pKMl (see Figure 6.4). Sequence data were obtained from the genomic insert of pMDl and these, like the sequence data from pKMl, were identical with the ORF YBR239c - 98.6% identity over 141bp. Additionally, each plasmid was transformed into the reciprocal strain and the phenotypes of the transformant strains were analysed. The thi2 complementing clone, pKMl, was found to confer thiamine prototrophy on the tyl-3 strain and pMDl was found to confer a TH12 phenotype on the thi2 strain. Therefore pKMl and pMDl must carry the same genomic insert.

Whether the two mutations were complemented by the same gene or adjacent genes within the genomic library clones was then investigated. The plasmid which did not rescue thi2 to thiamine prototrophy, TnpKMl, was transformed into the mutant strain Tyl-3 and the resultant strain was found to be thiamine auxotrophic. This indicated that thi2 and tyl-3 are allelic.

6.5.1 Segregation of thi2 and t y l - 3 .Since the complementing clones of thi2 and ty l-3 were identical, it would be

expected that the 058-M5 and tyl-3 mutations would be allelic. However a heterozygous diploid formed between these strains was prototrophic for thiamine (Chapter 3) suggesting that the mutations lie in different genes. This presented a paradox; segregation of the two alleles was therefore analysed. The diploid was sporulated and from nine tetrads analysed, all thirty six haploid progeny were found to be Thi' (data not shown); thus the thi2 and tyl-3 alleles segregated 2:2, revealing that they are very tightly linked.

6.5.2 Southern blot analysis of T yl-3.Southern blot analysis was carried out in order to investigate whether the Tyl-3

strain was disrupted at the TH12 locus by insertion of a Ty element. Genomic DNA was

71

MVNSKRQQRS K K VASSSK VP PTKGRTFTGC WACRFKKRRC DENRPICSLC AKHGDNCSYDIRLMWLEENI Y K V R K H SL IS SLQARKSKSK P L C Q K ISK SR FKQMTHFRQL S P P T S D C E D S

VHEASKETTL P N D N T F T IS V RRLKIYNNAV ASVFGSMTNR DYTQKRIDKK LDELLNMVEN

D IS V V N I.N C S KHGPYSVFRA N PA A V T SA L T DQLPSPGHSM SSA E E T T T A A L S S P P E D S T S

L I D I I Q G K I F GILWFNCYGN M ILNRQEYTT WFINKMRNSL T T E F IR F L G K IID D P D IN M A

S C L F K E C IA R WSCVDWQSIA IT M L V IIH G Y TCPNLTKLLR VWFLQQKLLR F SM Y PL V N FI

INNTQDLDVL YHCNGLLGNA DLFEDPYQDE LTSELH V LV T ERLVNSWKDT ILQQLCSCQD

TT L SC SQ L R Y WQLQLKCNQQ FYKDVYAMQD

Figure 6.3

Amino acid sequence of the Thi2p product.

The amino acid sequence of YBR240c was conceptually translated. The putative zinc finger motif is highlighted in the sequence.

Figure 6.4

Comparison of restriction maps.

The restriction maps of the DNA inserts carried by plasmids pKM l and pM Dl. The map for pKMl is derived from sequence data; the map for pMDl is derived from restriction mapping.X -Xhol, E - EcoKL, B - BamHL.

isolated from the wild-type strain W303, the Ty mutagenesis parent strain DG622, the thi2 strain 058-M 5, and the mutant Tyl-3. The DNAs were digested with the restriction endonuclease EcoRl, transferred to a nylon membrane and hybridised with a fluorescein labelled 3.5kb HindlU fragment from pKMl (Figure 6.5). A fragment of size approximately 1.14kb had hybridised to the probe within W303, DG622 and 058-M5 genomic DNAs; within the Tyl-3 DNA this fragment was not present, instead two ZscoRI fragments of size ~1.7kb and ~4kb hybridised to the probe. Therefore the TH12 locus in Tyl-3 had been disrupted, presumably by insertion of the Tyl element.

6.5.3 Crossing the th i2 disruption strain with t y l - 3 .Because the mutation in 058-M5 was likely to be a point mutations, as it was induced

by EMS mutagenesis, a missense or nonsense may have arisen. These may result in either a full length product carrying a mutation in an important domain or a truncated protein product, which although these were unable to carry out the designated function in a haploid strain, may have some function in a heterozygous diploid formed from a cross with Tyl-3, through intra-allelic complementation. It therefore seemed prudent to cross the Ty induced mutant, Tyl-3, against the disruption strain, in order to check for complementation. The resultant heterozygous diploid was found to be a thiamine auxotroph. This confirms that thi2 and tyl- 3 mutations affect the same gene i.e. they are allelic.

6.6 M olecular cloning of TH 13 .Complementing library clones of the thi3 strain, T49-2D, were isolated using the

YCp50 based yeast genomic DNA library. Seven of the resultant 6000 Ura+ transfomants were found to be thiamine prototrophs and these all reverted to thiamine auxotrophy when Ura' segregarits which had lost the library plasmids were tested. The library plasmids were isolated from the seven Thi+ transformants and were transformed back into T49-2D; 100% of the transformants displayed thiamine prototrophy, confirming that the plasmids carried the complementing Thi+ gene. Restriction analysis showed that the seven plasmid DNAs were identical; one of these was designated pKM2.

6.6.1 Analysis of pKM2Sequence data from the thi3 complementing clone pKM2 using the clockwise

oligonucleotide primer complementary to the DNA flanking the cloning site of the YCp50 vector, were obtained. These were found to be homologous to the ORF YDL085w (89.8% identity over 704bp), whilst the sequence data from the counter-clockwise primer showed identity to the ORF YDL079c (90.7% identity over 580bp). The restriction map and candidate ORFs within this region are shown in Figure 6.6.

72

1 2 3 4

A )~4kb

2.2kb

1.8 kb

1.14kb

THI2

2.193kb 1.137kb 1.775kbE coR l E coR l E coR l

H in& m H indU lA Hybridisation probe a

Figure 6.5.

Southern blots of THI2 locus in various strains.

Genomic DNAs were isolated from various yeast strains and digested with £coRL

Panel A) Autoradiograph of genomic DNAs hybridised with the fluorescein labelled 3.5kb H indH fragment, as shown.

Lane 1 - W303 DNA Lane 2 - DG622 DNA Lane 3 - 058-M5 DNA Lane 4 - Tyl-3 DNA

Panel B) Restriction map of the DNA surrounding the THI2 locus.

Figure 6.6

Restriction map and ORFs of pKM2.

The restriction map of the 8.9kb DNA insert of plasmid pKM2 and the candidate ORFs encoded by this DNA are displayed.The site of insertion of Tn1000::U RA3 fragment, the DNA fragment used for the hybridiastion probe and the site of excision for the disruption cassette are indicated by arrows.

YDL085w|- »

YDL084w

Hpal Clal Clal4 ------- -----------------

6.293kb

1.6 kb Hpal fragment used for hybridisation probe and gene disruption

< ►

YDL080cYDL079c

TClal Hpal Hpal. . . h >

1.602kb 0.33 kb

Site of insertion of Tnl000::URA3

6.6.2 Identifying the THI3 ORF.In order to locate the complementing ORF within the genomic insert the plasmid

pKM2 was subjected to Tn1000 based bacterial transposon mutagenesis (Sedgwick and Morgan, 1994). When the resultant Tn1000 disrupted pKM2 plasmids were transformed back into the thi3 mutant one plasmid failed to confer thiamine prototrophy; this was designated TnpKM2 (non-rescuer of thil). The complementing ORF of thi3 was identified by locating the site of insertion of the Tn1000 within TnpKM2. Sequence data was obtained (using the LTR primer, 5) from the ends of the TnlOOO insert in TnpKM2. These were

compared with sequences in the EMBL database and were found to be identical to an ORF of unknown function on chromosome IV, YDL080c. Thus it can be concluded that the thi3 mutation was complemented by YDL080c.

6.7 Disruption of the YDL080c locus.In order to show that YDL080c represented the THI3 wild-type gene and not a

suppressor, the locus was disrupted using the one-step gene disruption method. The TnpKM2 plasmid was digested with Hpal which released a fragment of approximately 7.5kb containing the entire Tnl000::URA3 sequence along with flanking DNA from the YDL080c locus (Figure 6.6). The DNA was isolated from an agarose gel and transformed into a THI3 yeast strain - W 303a. Transformants were selected on SD medium lacking uracil then four

of these were purified to single colonies and tested for thiamine auxotrophy. All four W303::Tnl000 colonies were found to be auxotrophic for thiamine suggesting that the THI3 gene had been disrupted.

Southern blot analysis was carried out to confirm that the YDL080c locus had been disrupted. Genomic DNAs isolated from the disruption strain W303::Tnl000 and the parent strain W303 were digested with H p a l , the fragments separated by agarose gel electrophoresis and transferred to a nylon membrane. Hybridisation using a fluorescein labelled 1.6kb Hpal fragment from plasmid pKM2 was carried out. A Hpal fragment of size 1.6kb was found to hybridise to the probe within the genomic DNA of W303 (Figure 6.7, lane 2) corresponding to an undisrupted YDL080c locus. However a ~8kb DNA fragment hybridised to the probe within the W303::Tnl000 DNA (lane 1), indicating that the YDL080c locus had been disrupted by the 7nl000::URA3 DNA fragment.

That the wild-type THI3 locus had been successfully disrupted was confirmed genetically by crossing the disruption strain, W303athi3::Tnl000, against the original thi3

mutant, T49-2D. The resulting heterozygous diploid was found to be auxotrophic for

thiamine.

73

1 2

------------► ~8kb Hpal fragment

1.6kb Hpal fragment

Figure 6.7

Southern blots of YDL080c disruption.

Genomic DNAs were isolated from wild-type (W303) and W 303::T n l000 strains and digested with Hpal.Lane 1 - W 303::Tnl000 DNA Lane 2 - W303 DNA

The 1.6kb H pal fragment from pKM2 was used as a hybridisation probe.

6.8 Thi3p is a PDC-like proteinSince YDL080c ORF had been confirmed as the THI3 gene, the nucleotide sequence

was translated, using the program Gene Jockey II (Biosoft). The deduced gene product was found to be a 610 amino acid protein. The S. cerevisiae genome sequencing project had recen tly been com pleted and this ORF had already been analysed (http://speedy.mips.biochem.mpg.de/mips/yeast/). It was found to be a putative pyruvate decarboxylase with homology to the S. cerevisiae PDC proteins Pdclp, Pdc5p and Pdc6p (see Figure 7.2, Chapter 7). It is known that PDC enzymes bind TPP as cofactor; interestingly, a TPP binding consensus sequence was also found within the Thi3p. The amino acid sequence with the putative TPP-binding site highlighted is shown in Figure 6.8.

6.9 Growth phenotypes of disruption strains and the original mutantsThe phenotype of the disruption strain and the original mutant were compared. The

wild-type strain (W303) was able to grow on minimal medium with and without thiamine; the thi3 strain (T49-2D) was able to grow on minmal medium supplemented with either thiamine or both precursors together - HMP+HMP, but not either of the precursors singly; the mutant strain Tyl-3 and the thi2 strain 058-M5 could only grow when thiamine was available (although there was some residual growth of 058-M5 when HMP+HET were provided together). This indicated that the Thi2p is required for activation of genes involved in downstream functions (THI6, for example) as well as genes acting in synthesis of the precursors (e.g. THI4 and THI5).

The thi3 strain, T49-2D, was able to grow in the presence of both the precursors, but not each separately, suggesting perhaps a requirement for the Thi3p of genes involved in upstream functions, but not necessarily for downstream ones.

6.10 D iscussionThe wild-type genes defined by the thi2 and thi3 mutations have been identified. The

THI2 gene is localised to the right arm of chromosome II, YBR240c; it encodes a putative transcriptional activator with a DNA binding 'zinc finger' motif, indicating that Thi2p may activate the transcription of thiamine genes by binding directly to their promoters. When the amino-acid sequence is compared to other transcriptional activator proteins with zinc-finger motifs, such as Gal4p there is little other sequence identity, suggesting perhaps that Thi2p may bind specifically to thiamine promoters.

The Ty induced mutation tyl-3 has been shown to be allelic with thi2 by the 2:2 segregation of the alleles resulting from tetrad analysis. Also it was shown that the Tyl-3 strain had been disrupted at the THI2 locus by a Ty insertion. That 058-M5 and Tyl-3 were able to complement each other was therefore unexpected and suggested that the THI2 gene

74

Pyrimidine binding domain

MNSSYTQRYA LPKCIAISDY LFHRLNQLNI HTIFGLSGEF SMPLLDKLYN IPNLRWAGNS NELNAAYAAD

GYSRLKGLGC LITTFGVGEL SAINGVAGSY AEHVGILHIV GMPPTSAQTK

Pyrophosphate binding domain

QLLLHHTLGN GDFTVFHRIA SDVACYTTLI IDSELCADEV DKCIKKAWIE QRPVYMGMPV NQVNLPIESA

RLNTPLDLQL HKNDPDVEKE V IS R IL S F IY KSQNPAIIVD ACTSRQNLIE ETKELCNRLK FPVFVTPMGK

GTVNETDPQF GGVFTGSISA PEVREWDFA DFIIVIGCML SEFSTSTFHF QYKTKNCALL YSTSVKLKNA

TYPDLSIKLL LQKILANI.DE SKLSYQPSEQ PSMMVPRPYP AGNVLLRQEW VWNEISHWFQ PGDIIITETG

ASAFGVNQTR FPVNTLGISQ ALWGSVGYTM GACLGAEFAV QEINKDKFPA TKHRVILFMG DGAFQLTVQE

LSTIVKWGLT PYIFVMNNOG YSVDRFLHHR SDASYYDIQP WNYLGLLRVF GCTNYETKKI ITVGEFRSMI

SDPNFATNDK IRMIEIMLPP RDVPQALLDR WVVEKEQSKQ VQEENENSSA VNTPTPEFQP LLKKNQVGY

Figure 6.8

Amino acid sequence of the THI3 ORF.

The amino acid sequence of YDLO80c was conceptually translated. Residues conserved amongst TPP-binding proteins are highlighted in the sequence and the putative TPP binding consensus motif from Hawkins etal. (1989) is underlined.

product is bifunctional and that the mutants were each defective in the one of these functions allowing intra-allelic complementation to occur. The finding that the thi2::Tnl000 disruption strain did not complement ty l-3 seems to support this idea and implies that intra-allelic complementation had occurred in the heterozygous diploid produced from the cross of Tyl-3 with 058-M5. The Thi2p product may dimerise and have two domains, one required for DNA binding and the other one for activation perhaps mediated through a protein-protein interaction.

The THI3 gene was localised to the left arm of chromosome IV, YDL080c. It encodes a protein that is homologous to PDC structural genes and contains a putative TPP binding domain. Thus the Thi3p may be the sensor of internal TPP concentrations, mediating its effect by the activation or deactivation of thiamine genes.

Although the genes THI4, THI5 and TH112 like PH03, THI6 and THI80 require the functions of the THI2 and THI3 genes for their activation, supporting the hypothesis for thiamine regulation (Nishimura et a i, 1992b), the observation that thi3 mutants, including the disruption strain, were able to grow on minimal medium lacking thiamine when both precursors were provided, suggested that there must be some activity of the enzymes which convert HMP and HET to TPP, including the gene products of THI6 and THI80. Thus there may be a basal level of expression from these, and other, downstream thiamine genes which does not require activation by Thi3p.

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CHAPTER SEVEN

General discussion

The work carried out here has shown that mutant strains displaying thiamine auxotrophic phenotypes can arise for a number of reasons (i) through mutations in thiamine biosynthetic genes (ii) through mutations in TPP-dependent enzymes (iii) through mutations in regulatory functions.

7.1 Auxotrophic mutations in biosynthetic genesAmongst a collection of new UV and Ty induced thiamine auxotrophic yeast mutants

I isolated a number in the thiamine biosynthetic genes, THI4, and THI6, as described in Chapters 3 and 4. The number of thi4 mutations far exceeded those found in any other gene; these totalled 141. Although some of these could have been duplicates of the same mutational event, a minimum of four mutations had arisen independently. It is interesting that none of these thi4 mutations arose from UV mutagenesis. That so many Ty inserions had occurred within this gene suggests it to be a hotspot for this type of event. Ty retrotransposition has been shown to be influenced by the presence of tRNA genes and hence Ty elements may be targetted to loci containing tRNA genes (Hani & Feldmann, 1998). In more than a hundred Tyl insertions into chromosome III most were clustered upstream of tRNA genes and 57% were within 400bp of a tRNA gene, independent of nucleotide sequence at the target site (Ji et al., 1993). Also it has been found that pre-existing Tyl elements show affinity for tRNA genes (Oliver et al., 1992). This suggests a possible interaction between the Tyl integration apparatus and RNA PolIII transcription machinery or the chromatin structure upstream of PolIII transcribed genes. A tRNA-gly (UCC) gene is located just upstream from the THI4 gene, between nucleotides -782 and -710 of the 5' non-coding region (Praekelt and Meacock, 1992). Interestingly, just upstream from this tRNA gene are five 5 sequences from

Tyl elements, presumably left behind after transposition and recombinational excision. The presence of this tRNA gene and 5 sequences could explain why so many mutations, arising

from Ty insertions into the THI4 gene (or its promoter), were recovered.Two thiamine auxotrophic mutations were isolated which mapped to the THI6 gene;

one arose following UV mutagenesis (UV4) and one through Ty insertion mutagenesis (Ty3-1). The TH16 gene encodes a bifunctional enzyme comprising the two activities of HET kinase and TMP-PPase (Kawasaki, 1993; Nosaka et al., 1994). Why these two thiamine biosynthetic functions should be combined into a single protein could be evolutionarily significant, especially since it appears to carry out an essential (TMP-PPase) and a non-essential (HET kinase) function. Has it occurred accidently or in order to enhance the stability and/or efficiency of the enzymes? For example once HET is phosphorylated it

76

may be retained by the enzyme until HMP-PP becomes available so that TMP can be synthesised quickly and efficiently. Intriguingly, the two functions are encoded by distinct genes in E. coli, namely thiB and thiE (TMP-PPase) and thiM (HET-kinase) (Mizote & Nakayama, 1989; Begley, 1996).

No other mutations of biosynthetic genes were obtained from either the UV or Ty insertion mutagenesis screens suggesting two possible scenerios. Firstly, the pathway genes have not been saturated with mutations which is possible as the analysis revealed that most of the mutants fell into one complementation group, thi4, whist the other groups were represented by only one mutant. Secondly, that mutations in other biosynthetic genes were not recoverable by the screening strategies employed here. This is illustrated by the recovery of the pdc2 mutants, uv2 and uv3 (described in Chapter 3). Although these strains were isolated as thiamine auxotrophs both exhibited pronounced growth defects which could easily have resulted in them being overlooked; the growth defects were later identified as being due to an inability to metabolise glucose, which was the sole carbon source in selective media used. Thus other potential thiamine auxotrophs may have been lost during the screening procedures due to unforeseen factors which were not compensated for e.g. the mutated proteins are also involved in other metabolic pathways.

7.2 Mutation in a TPP-dependent enzymeA mutation in a TPP-dependent enzyme manifested itself in a thiamine auxotrophic

phenotype. In Chapter Five it was found that the first documented thiamine auxotroph, thil, was mutated in a gene involved in isoleucine and valine biosynthesis, ILV2. The gene product, AH AS, has homology to other TPP-requiring enzymes, especially POX. It contains a consensus TPP binding motif, GDGX[2 5-2 8 ]NN, the residues of which are primarily concerned with binding the pyrophosphate end of the TPP molecule through interactions with a divalent cation. Experiments investigating the cofactor-binding site of PDC from Z. mobilis by site-directed mutagenesis have revealed that some mutations in Asp440 or Asn467 (D and N, found at the ends of the motif) affect the binding of TPP and the divalent cation without affecting the activity or binding of the substrate (Candy and Ronald, 1994). An Asn467Asp substitution resulted in an active enzyme whilst a Asn467Gln mutation caused a total loss of TPP binding and activity. Also an A sp ^ G lu substitution resulted in an active enzyme with a much reduced affinity for TPP. These results implied that the size of the amino acid side chain is crucial in the TPP-binding site, since the additional methylene group in a Gin compared with a Asn or a Glu compared with a Asp residue would push the amino acid side chain further into the coenzyme-binding pocket. The thil allele encodes an AHAS with a Asp176—»Glu substitution, which introduces an additional methylene group to the

local environment. Although this Asp residue does not form part of the consensus TPP- binding motif it may nonetheless form part of the fold where the cofactor binds. Whatever its

77

role is, it has an effect on TPP-binding to AHAS and therefore must be involved in the enzyme-cofactor interaction either directly or indirectly.

During the characterisation of the thil strain the true phenotype was revealed to be one of thiamine o r isoleucine and valine auxotrophy, due to a mutation which lead to a reduced affinity of the enzyme for TPP. Only when the strain was given additional thiamine was the enzyme able to carry out its function, allowing the synthesis of isoleucine and valine. This unusual phenotype may be of use to isolate yeast strains which overproduce TPP. If the thil strain was exposed to a mutagen, perhaps a chemical mutagen such as EMS, and then grown on medium deficient in thiamine and isoleucine and valine, the only strains able to grow should either have a fully functional AHAS enzyme, through reversion of the th il mutation, or have an elevated intracellular TPP concentration. It would be simple to eliminate back mutations since the thil mutation produces an additional site for the restriction endonuclease, Stul. Therefore any strains able to grow would be subjected to colony PCR and the products digested with Stul. Strains which still retained the thil mutation could then be subjected to intracellular thiamine assays and these levels compared to wild-type cells.

Another application for this proposed reduced TTP-binding phenotype of Hv2-thilp could be in brewing. One of the products of AHAS action, acetolactate, can be converted non-enzym ically to diacetyl, the substance responsible for the off-flavour in beer (Gjermansen et al. , 1988). Therefore since ALS assays showed that the Ilv2-thilp had a reduced ALS activity (Figure 5.7, Chapter 5), use of the th il mutant yeast strain in fermentation may be helpful in reducing the build up of diacetyl and the likelihood of off- flavour beer.

The finding that addition of the thiamine precursor, hydroxy methyl pyrimidine, to the medium also rescued the thil defect (Chapter 3, Table 3.3) is curious. In this study there are other examples of "thiamine auxotrophies" which are rescued by the addition of the pyrimidine precursor e.g: a mutant of the PDC2 gene, uv3, that could grow in the presence of HMP, as well as the Ty 1-128 strain described in Chapter 6 which was similarly restored to growth and yet was found to be allelic with thi3. This phenomenon may be indicative of pyrimidine synthesis being the rate limiting step in the production of thiamine, such that addition of this precursor to the medium allows the cell to take it up and combine it with HET-P so relieving the thiamine defect. This scenario would assume that the cell is not restricted in availability of the thiazole precursor. Why the two precursor pathways should be differentially regulated perhaps reflects the alternative functions of some of the gene products. For example, we know that the Thi4p is not only involved in synthesis of HET but is also implicated in mitochondrial DNA damage repair (Machado et a l, 1997).

HMP is taken up via an active transport system in common with thiamine (Iwashima et al., 1990b) but in contrast to thiamine, uptake of HMP displayed counterflow efflux whereby accumulated HMP was released form the cells. This overshoot phenomenon may be a mechanism by which yeast cells rid themselves of excess HMP. By contrast HET is

78

taken up via facilicated or simple diffusion and is rapidly converted to HET-P, effecting metabolic trapping (Iwashima et al., 1986). This observation could mean that build up of HMP, or a phosphorylated derivative, is toxic to the cells. It could also account for the apparent 'bottleneck' in HMP synthesis that was observed in a number of mutants i.e. there may be a ready supply of the thiazole precursor in cells (in the form of HET-P) from uptake of the compound and/or salvage synthesis from thiamine followed by phosphorylation (metabolic trapping) whilst HMP is lost from the cell. Therefore the mutants which display HMP auxotrophy may do so not because they are defective in the synthesis of this precursor (and indeed it has been shown that at least two of them are not) but simply because they require thiamine to be made and HMP is the limiting factor.

7.3 M utations in regulatory genesThe regulatory mutants, thi2 and thi3, were complemented using a low-copy plasmid

library and their respective wild-type genes were identified. The deduced amino acid sequence of Thi2p product implicated it as a DNA-binding transcription factor, since it contains a zinc finger motif similar to the transcription factor Gal4p. Although very little homology was found throughout most of the length of the two sequences there was one highly conserved domain, which corresponds to the Zn2-Cys6 "zinc finger" consensus

pattern (shown in Figure 7.1).The Gal4p transcription factor is a 881aa protein which binds to the 17bp

symmetrical U A S g a l sequence as a dimer (Marmorstein et al., 1992). The protein comprises a DNA binding region (l-65aa), residues involved in dimerisation (65-94), a weak dimerisation region (50-65), three acidic activating domains (aa 94-106, 148-196 and 768-881) and an inhibitory region at the carboxy terminus which binds Gal80p. If Thi2p behaves like Gal4p, binding to DNA as a dimer and having different domains for binding, dimerisation and activation or protein-protein interaction, then this may explain how it was possible for two thi2 mutant strains, 058-M5 and Tyl-3, to complement each other. Additionally, since Tyl-3 belongs to a complementation group with 27 members (Chapter 3) it might be interesting to cross some of these other strains with 058-M5 to see if some or all of them are able to complement the thi2 mutation.

The consensus binding sequence of Gal4p comprises CGG X [H ] CCG; similar

DNA-binding proteins, such as Lac9p (from K. lactis) and Pprlp (from S cerevisiae) also bind to sites comprising a palindromic CCG triplet. Although the Thi2p shares a CX[2 ]CX[6 ]C motif with these proteins it has few other regions homologous with Gal4p,

and inspection of the promoters of thiamine regulatable genes which are thought to be under the positive regulation of Thi2p (P H 03 , 777/4, 777/6, THI80 and the THI5 gene family) reveals no similar binding sites. Potential binding sites for Thi2p may be identified in various ways. Firstly, general thiamine regulated UAS regions may by identified by carrying out

79

10 20 30 40 50 60 70I I I I I I I

THI2 MVNSKROORSKKVASGSKVPPTKGRTFTGCWACRFKKRRCDENRPICSLCAKHGDNCSY----------------------------------- DIRLMWLEEN IYKVRKHS• • | • | • | | • • | • • | I• • I • • I • • I *1

GAL4 M---------------------KLLSSIE-------------------OACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDLDI I I I I I I10 20 30 40 50 60 70

Figure 7.1

Comparison of Thi2p and Gal4p amino acid sequences.Homology search carried out on the amino acid sequences of Thi2p and Gal4p, using the software Gene Jockey II. Underlined is the consensus sequence for proteins containing the "zinc finger" motif (obtained from MIPS, http://speedy.mips.biochem.mpg.de/mips/yeast/).

promoter deletion analyses of the THI genes and assaying for expression, either through use of THI-LACZ reporter gene constructs, or through Northern blot analyses. Secondly, DNA fragments containing any putative sequences could be subject to gel retardation assays, using cell extracts of Athi2 and THI2 strains of yeast. Thirdly, if the Thi2p was purified then

footprinting analysis would identify any region of DNA protected by this putative DNA- binding protein. The latter two methods would not only establish whether the protein did in fact bind DNA, but would also identify a Thi2p binding site.

The deduced amino acid sequence of the Thi3p product was found to be homologous to those of the PDC structural proteins - Pdclp, Pdc5p and Pdc6p. Figure 7.2 shows the results of a homology search between these sequences. All of these proteins share a conserved domain which is found in all TPP-binding enzymes, the GDGX(2 5 -2 8 )NN consensus motif. The function of Thi3p may therefore be to bind TPP and act as the "sensor" of TPP levels within the cell such that when TPP is bound the Thi3p-TPP complex destabilises the transcription initiation complex of THI promoters leading to deactivation (repression) of thiamine-regulated genes.

How the third known thiamine activator, Pdc2p, may effect regulation at the THI promoters is less clear. This protein contains an asparagine rich box (ARB) which appears to be a transcriptional activation domain, since fusion of this fragment with the Gal4p DNA binding domain was able to activate transcription from the GAL1 promoter (Raghuram et al., 1994). The ARB is conserved in Drosophila melanogaster transcription factors, such as mastermind, caudal and cut, suggesting a similar and important function for Pdc2p - transcriptional activation. The ARB is also found in the negative regulator of Ras proteins, Rpilp, indicating that Pdc2p may be involved in protein - protein interaction rather than protein - DNA. Figure 7.3 shows the possible interplay of the elements involved thiamine regulation. The Thi2p binds to a UAS of the THI promoter whilst Pdc2p recruits RNA PolII to the TATA box and Thi3p stabilises the initiation complex, allowing transcription to occur (Panel A). When TPP concentration increases either by synthesis or transport, it enters the nucleus and is bound by Thi3p (Panel B). This causes the protein to lose its conformation such that it is unable to bind the initiation complex and the components fall apart, hence transcription is deactivated (repressed) (Panel C).

Now that the THI2 and THI3 genes have been isolated it would be possible to determine whether any of the det mutations isolated by R. Burrows, which lead to derepressed expression of TH14 in the presence of thiamine, are allelic with either of these regulatory genes. It has already been shown that they are not allelic with the other regulatory

gene, PDC2 (Burrows, 1997).

80

Figure 7.2

Alignment of the amino acid sequences of Thi3p and the PDC structural proteins.

Homology search was carried out on the predicted amino acid sequences of Pdclp, Pdc5p, Pdc6p and Thi3p using the software Gene Jockey II. Underlined is the consensus sequence for proteins containing the “TPP- binding” motif, described by Hawkins et al. (1989).• indicates identity I indicates conserved substitution

CytosolNucleus

TranscriptionPDC2

THI2 RNApol THIUAS TATA

TPP TPPTPP TPP TPP

/ Cytosol/

/Nucleus

//

Transcription---------

UAS

RNApdlTATA

THI

TPP TPPTPP TPP Cytosol

NucleusTPP

THI3 RNApol — -- NO Transcription(Tffl2) (PDC2) 1 X ^

________________________________ 1 THIUAS TATA

Figure 7.3

Model for thiamine gene regulation.

Taken from Burrows (1997).

7.3.1 The YDL080c ORFAnother role for the YDL080c ORF has recently been uncovered. During a

biochemical study of the metabolism of leucine and its possible routes to isoamyl alcohol (IAA) the putative pyruvate decarboxylase activity of the product of this gene was implicated (Dickinson et al., 1997). Figure 7.4 shows a schematic plan for how leucine is metabolised.

The authors suggested four possible routes for the catabolism of leucine to IAA; all of these involved the initial deamination of the amino acid to a-ketoisocaproate (a-KIC)

catalysed by the branched-chain amino acid transaminase (BCAAT). From here four possible routes were open: first to isovaleryl CoA via catalysis of the branched chain a-keto acid

dehydrogenase (BCKD) and then to isovaleric acid via catalysis of the acyl CoA hydrolase (ACH); secondly, directly to IAA via action of pyruvate decarboxylase; thirdly, to a - hydroxyisocaproate (a-HIC) via an a-ketoisocaproate reductase; finally, directly to IAA via

a pyruvate decarboxylase-like enzyme. The first three possibilities were found to not encompass the major catabolic route to IAA for the following reasons: a mutant that lacked BCKD activity (which also lacked PDH, KGD, glycine carboxylase and lipoamide dehydrogenase due to a disruption in the gene LPD1 gene) was still able to produce IAA and did not contain isovaleryl CoA, when cells were grown on glucose; a triple disruptant pdcl, pdc5 , pdc6 completely lacking PDC activity was still able to produce IAA when grown on ethanol as a carbon source; a-H IC was not converted to IAA when cells were grown in

medium in ethanol with leucine as the sole nitrogen source, although it had been demonstrated that S. cerevisiae is able to convert a-KIC to a-HIC and three ORFs had been found in the genome which could putatively encode an a-ketoisocaproate reductase enzyme.

That the product of the YDL080c gene might be responsible for the major catabolic route of leucine to IAA was demonstrated by assays involving two strains carrying disruptions at this locus. These gave a much reduced level of IAA however there was still a significant amount of IAA present, indicating that although YDL080c encodes the major a-KIC decarboxylase,

at least one other enzyme is capable of catalysing this reaction. Indeed, with a quadruple disruptant of p d c l , pdc5 , pdc6 and ydl080c the strain gave a similar level of IAA to the single ydl080c strain revealing that these enzymes did not carry out this function. Whether the other two ORFs found in the S. cerevisiae genome which also display "PDC-like" characterisitics, YDR380w and YEL020c, are able to carry out this function has not yet been investigated.

It therefore appears that Thi3p has an enzymatic function whilst also being a transcriptional activator of thiamine genes which binds the endproduct of the pathway. There are other examples of such proteins with dual functions in catalysis and regulation e.g. Pdclp, which also has PDC activity and yet is able to regulate expression of the PDC5 gene (Hohmann etal., 1996)

81

LEUCINE a-KICBCAAT

PDC /B

PDC-like /Da-HIC

IAA

---------- ► ISOVALERYLBCKD * COA

ACH

ISOVALERIC ACID

Figure 7.4

Potential metabolic routes for the catabolism of leucine to IAA.

Four possible pathways were proposed for the catabolic routes to IAA (adapted from Dickinson et al., 1997).BCAAT denotes branched-chain amino acid transaminase; BCKD

denotes branced-chain a-keto acid dehydrogenase; ACH denotes acyl

CoA hydrolase; PDC denotes pyruvate decarboxylase.

7.4 Future workFuture work could be carried out on the thil mutant strain, firstly, in order to confirm

biochemically that the phenotype observed, i.e. thiamine auxotrophy in the absence of isoleucine and valine, was indeed due to a reduced binding affinity of the mutant AHAS for the cofactor TPP. This would involve carrying out enzyme assays on cell-free extracts of the strains carrying the various 1LV2 alleles, or by purifying the enzymes. However this may prove difficult because AHAS is localised in the mitochondria and also because the enzyme is labile and present in low abundance (Poulsen and Stougaard, 1989). As mentioned previously this strain may also be used to try and isolate mutations which result in an increased intracellular TPP concentration (Section 7.2).

Whether Thi2p is a bona fide DNA binding protein could be tested in two ways (as discussed in Section 7.3): any putative thiamine regulatory sequences may be subjected to gel retardation assays, using cell extracts of Athi2 and THI2 strains of yeast; and

footprinting analysis could be used to identify any region of DNA protected by purified Thi2p.

The mechanism by which THI3 affects regulatory transcription of thiamine genes could be mediated through protein - protein interactions of Thi3p or through the product of its catalytic function i.e. IAA. It should be simple to test whether IAA itself has an effect on thiamine gene expression by adding it to thi3A cells and testing for alleviation of the mutant

phenotype by assaying growth on thiamine deficient medium. If it seems that it is the protein itself which is required for thiamine gene activation then protein - protein interactions with Thi2p, Pdc2p and any other potential activating proteins could be tested biochemically via affinity-tagging one of the proteins and then carrying out affinity chromatography. Alternatively, if antibodies were available immuno-coprecipitation could be carried out. The interacting protein could then be identified either by Western blotting analysis or via protein sequencing. Additionally the two-hybrid screen could be used to identify any other potential interacting proteins.

Once the functions and interactions of Thi2p, Thi3p and Pdc2p have been established it would be of interest to investigate the nature of the original thi2 and thi3 mutations in strains 058-M5 and T49-2D since these probably carry point mutations. This could be carried out in the same way that the thil mutation was identified in Chapter 5 i.e. by isolation of the mutant allele via gap repair followed by DNA-sequencing. This may then be useful in identifying residues which are important in protein - protein interactions, or in the case of Thi2p, in DNA-binding.

Since no new thiamine biosynthetic genes were identified in this study, an alternative way to isolate them may be approached using the Saccharomyces Genome Database, and searching for genes which carry promoters with thiamine regulatory domains. Alternatively differential cDNA screens may uncover novel thiamine regulated genes.

82

From all of these studies it is clear that a lot of work still remains to be done in order to fully understand the genetic and molecular mechanisms underpinning thiamine metabolism and its involvement in intermediary metabolism. The full picture of how this vitamin is synthesised and regulated in microorganisms is still emerging.

83

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